DE102004031116A1 - Method for finding pain-relevant substances using pain-relevant proteins - Google Patents

Abstract

The invention relates to a method for finding pain relevant substances using the proteins KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and the use of the compounds identified in the medicaments and diagnostics by the method according to the invention the pain therapy. The invention further relates to the novel nucleic acid and amino acid total sequences of the mouse BAB31214 protein as well as the nucleic acid and amino acid partial sequences of the human KIAA0378 protein and the rat PID12832121 protein.

Description

The The invention relates to a method for finding pain relevant Substances using the proteins KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and the use of the identified by the inventive method Compounds in medicines and diagnostics as well as in pain therapy. The invention further relates to the novel nucleic acid and amino acid total sequence, of the mouse BAB31214 protein as well as the nucleic acid and amino acid partial sequences human KIAA0378 protein and rat PID12832121 protein.

in the Prior art are different for the therapy of pain Drugs, e.g. Acetylsalicylic acid, paracetamol, dipyrone, Tramadol, morphine and fentanyl are available. In addition, there are also substances such as amitryptiline and ketamine, for the treatment of pain patients for use. The treatment of pain is becoming increasingly important so that the regimens of pain, especially chronic Pain, be continuously refined.

So The pain research of recent years brought the basic Recognizing that the development of chronic pain in particular is plastic changes of the nervous system, especially in the nociceptive neurons of the Posterior root ganglia and neurons in the dorsal horns of the Backmarks, underlying (as an overview see: Coderre et al. 1993; Zimmermann & Herdegen, 1996). The neuronal plasticity goes hand in hand with changes in the expression of certain genes and leads to long-lasting change of the phenotype the affected neurons. The concept of neural plasticity became so far mainly on development, learning and regeneration processes applied. However, recent findings from pain research show that this concept also applies to pathophysiological processes (Tölle, 1997).

• – erhöhte Empfindlichkeit und verringerte Reizschwelle peripherer Nozizeptoren,
• – Aktivierung so genannter stiller Nozizeptoren,
• – Reorganisation rezeptiver Felder,
• – Erregbarkeitszunahme im Rückenmark

The chronification of pain is already relatively well characterized in animal experiments on the phenomenological level. The induction of chronic pain states leads in particular to the following changes:

• Increased sensitivity and reduced stimulation threshold of peripheral nociceptors,
• - activation of so-called silent nociceptors,
• - Reorganization of receptive fields,
• - increased excitability in the spinal cord

In Pain perception comes next to the nociceptors, as the primary sensory Afferents, including the downstream neurons in the dorsal horn of the Backmarks a special meaning too. A chronification of the pain takes place by the excitability increase of the downstream neurons in the spinal cord This increase in excitability is also called "central sensitization" will the transmission of signals (electrical impulses) from the nociceptors to the Downstream neurons by inhibitory or excitatory Interneurone modulated. This changed signal transmission will also be a part of the chronification or the failure conventional Attributed to analgesics.

The dorsal spinal cord thus represents one of the most important switching points of the nociceptive Neurotransmission. Here nociceptive signals are both processed as well as modulated. The subcellular compartment of the involved Neurons that are listed above Pain-related processes The most important role is played by the pre- and postsynaptic membrane Here becomes presynaptic the transmitter exchange regulates and postsynaptically detects the neurotransmitters, resulting in activation or inhibition of the corresponding neuron leads. These functions are primarily of the synaptic membrane proteins dorsal spinal cord neurons exercised.

In spite of However, progressively more advanced therapies can be used in chronic pain conditions often no lasting improvement is achieved for the patient. Among others are for this the permanent changes involved nerve cells cause. In such permanent changes of nerve cells are proteins, in particular the mentioned synaptic Membrane proteins of the dorsal spinal cord, involved, their identification and characterization in the state The technique has been insufficient so far. Such proteins play an important role as molecular targets for the development of new analgesics and in particular for corresponding screening methods, i. for procedures that use substances be identified, which are used as analgesics can.

Of the The present invention is therefore based on the object, on the one hand Identify proteins that play a role in chronic pain conditions, and on the other hand to provide a method with which Use of these identified proteins is potentially pain-regulating Substances are to be found.

to solution the task of the invention was therefore first using selective protein-chemical methods a selective Analysis of the dorsal spinal cord tissue, more precisely in the dorsal spinal cord neurons, made localized synaptic membrane proteins. Based on the following These studies have been characterized as proteins that are known in the pain regulation are involved and thus new targets for the treatment of pain, especially chronic pain. In detail, the separation of subcellular fractions (myelin, synaptic Membranes, light membranes, etc.) using two-dimensional 16-BAC / SDS-PAGE as one for integral membrane proteins made compatible separation system and Based on the results, a proteomic map of the dorsal spinal cord created. Starting from synaptic membranes could be named after Separation in the two-dimensional 16-BAC SDS-PAGE identified a total of 55 proteins become. Of these, in particular according to the invention relevant Proteins further characterized.

According to the invention can such identified proteins are used in a process can be found with the pain-regulating substances, the applied in the therapy of especially chronic pain can be.

• (a) Inkubation einer zu testenden Substanz unter geeigneten Bedingungen mit einer Zelle und/oder einer Präparation aus einer solchen Zelle, die mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL und/oder mindestens ein Protein umfassend eine Aminosäuresequenz gemäß einer der 11, 18, 20, 27, 29, 31, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 und/oder mindestens ein Protein, für das eine Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 codiert, und/oder ein Protein, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 oder deren Antisense Nukleinsäure hybridisiert oder ein funktionelles Fragment oder eine funktionelle Variante eines der vorgenannten Protein synthetisiert hat,
• (b) Messung der Bindung der Testsubstanz an das mindestens eine von der Zelle synthetisierte Protein oder funktionelle Fragment oder funktionelle Derivat hiervon oder Messung mindestens eines der durch die Bindung der Testsubstanz an das Protein oder funktionelle Fragment oder funktionelle Derivat hiervon veränderten funktionellen Parameters.

Thus, a first aspect of the present invention is a method for finding pain regulating substances which comprises the following steps:

• (a) incubation of a substance to be tested under suitable conditions with a cell and / or a preparation from such a cell which comprises at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 encoded, and / or a protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense has hybridized nucleic acid or synthesized a functional fragment or a functional variant of one of the aforementioned proteins,
• (b) measuring the binding of the test substance to the at least one cell synthesized protein or functional fragment or functional derivative thereof or measuring at least one of the functional parameter altered by the binding of the test substance to the protein or functional fragment or functional derivative thereof.

The inventive method based on the fact that the potential pain-regulating effectiveness a substance over the interaction of this substance with a pain-regulated Protein or a protein with a pain-relevant distribution in the central nervous system (CNS), as described according to the invention can be identified.

"Pain-regulating" is a substance in the sense of this invention, if they have a potential regulating Influences the physiological pain event. Especially this regulatory influence is an analgesic effect. Under a "substance" according to the invention is a chemical Understood compound that potentially have an effect in the body can, in particular any compound suitable as a drug.

These include, for example, low molecular weight drugs, nucleic acids, fats, sugars, peptides (polypeptides) or proteins, antibodies. According to the invention, "low molecular weight" is a molecule having a molecular weight <2 kDa. An "active substance" within the meaning of the invention is a compound which causes an alteration, in particular a curative effect, in an organism to which it has been administered. These are preferably organically-chemically synthesized molecules, in particular molecules which are linked to the process proteins and polypeptides (in the present case the name for a protein or polypeptide which is present in the inventive method can be used) bind.

A "to be tested Substance "or" test substance "according to the invention is a substance as defined above, for which a potential pain-regulating Effect is assumed Such a test substance is with a cell of the invention or a preparation from such a cell is incubated under appropriate conditions, i. Test substance and cell are in an aqueous medium for a certain period (incubation period) reacted. The Incubation period is dependent on the nature of the substance and the associated interaction with a process protein. It can last a few seconds to several Hours, preferably amounts to between 1 minute and 60 minutes. The aqueous medium may preferably between 4 ° C and 40 ° C be tempered, preferably the temperature is at room temperature or at 37 ° C. Such tempering of the aqueous Medium can, for example, in an incubator or water bath respectively. Preferably contains the watery Medium suitable salts and / or buffer systems containing the aqueous medium at a desired pH, which is preferably between pH 6 and pH 8, preferably pH 7.0 and pH 7.5. Other substances, such as Coenzymes and Nutrient, can the aqueous Medium added become. Those skilled in the art will find such suitable incubation conditions and their variations well known. In general, these are physiological Conditions (for example 37 ° C, pH 7.2) or conditions that provide optimal measurement according to the method of the invention enable.

Under A "cell" according to the invention becomes a cell understood that a process protein or a protein according to the invention, as defined below, or a functional fragment or synthesized a functional variant of such a protein. Here they can be cells that are a process protein or protein of the invention expressing endogenously or to cells that are genetically engineered and thus a process protein or protein of the invention express. Cells according to the invention can come from immortalized cell lines or from native tissue. The Cell can be cultured separately or part of a tissue, in particular, of an organ in which the cell is isolated or still present in the cell structure. Preferably, such Cell, however, isolated from the tissue and dissolved the cell structure.

A "preparation" from a cell according to the invention provides by chemical, biological, mechanical or physical Treatment prepared preparation wherein such treatment alters the cell structure. at such preparations they are, for example, membrane fragments, isolated compartments the cell, isolated cytosol, tissue derived homogenate or a suspension of isolated cell organelles.

at one in the method according to the invention usable "protein" is a protein from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL selected is. These proteins are represented by the listed amino acids or nucleic acids coded. The aforementioned proteins are proteins, identified as pain-regulated or pain-relevant were. As already mentioned, this was a proteomic map of the dorsal spinal cord of an animal, in particular a rat, by the separation of subcellular fractions, such as myelin, synaptic membranes, light membranes, etc., followed by identification created by membrane proteins. From a total of 55 identified Proteins were particularly relevant to the abovementioned invention Proteins closer analyzed. This was initially a change The expression pattern is examined by expressing the proteins in an animal in which pain has been triggered, with a control animal, that does not cause pain activities was compared. Furthermore, in in situ hybridization experiments the cellular Localization of mRNA transcripts of the aforementioned invention relevant Proteins examined. Due to the investigations made a changed one Expression or a pain-relevant distribution of the aforementioned according to the invention relevant proteins found in pain-induced animals. The aforementioned proteins KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL are hereinafter referred to as "in the inventive method usable proteins "or" in the inventive method usable proteins "or Also referred to as "process proteins of the invention." For the inventive method it is irrelevant from which species the process proteins originate however, it is preferred to use the human, mouse or rat variant.

following become the process proteins of the invention described in more detail:

KIAA0378

This is the human protein KIAA0378, which is registered in the database as sp: 015083 or embl: AB002376. This protein is uncharacterized in the database and registered with an incomplete sequence. According to the invention, it has been possible to identify the missing N-terminal amino acid sequence and the associated nucleic acid sequence. The completed amino acid sequence now contains 149 additional amino acid residues at the N-terminus (see 11 ). The corresponding nucleic acid sequence is in 12 shown.

KIAA0378 consists mainly of an extended coiled-coil region, which presumably plays a role in the oligomerization of the protein. Only the first 140 amino acid residues are not present in a coiled-coil structure. The protein has neither a signal nor a transmembrane sequence (see 11 and 12 ).

According to the invention, a specific expression pattern of KIAA0378 could be detected for the first time, but not in the spinal cord (see 13 ). It shows that the KIAA0378 protein isolated from synaptic fractions of the dorsal horn is derived, at least in large part, if not exclusively, from primarily sensory neurons that terminate in the dorsal horn of the spinal cord. This selective expression of KIAA0378 clearly indicates that KIAA0378 in synaptic spinal cord endings is relevant for sensory signal transduction in the posterior dorsal horn. It can therefore be assumed that KIAA0378 has an important presynaptic function in the neurotransmission of primulatory neurons to neurons in the dorsal horn of the spinal cord. Since virtually all DRGs express neurons KIAA0378, it can be assumed that nociceptive neurons of the C- and A-delta fiber class KIAA0378 express as well, from which the pain-relevant role of KIAA0378 in nociceptive neurons can be deduced. For example, administration of KIAA0378 inhibitors close to the spinal cord could interrupt a "circulus vitiosus" of the spinal pain memory.

BAB31214 / Q9BU64

BAB31214 is the murine orthologue to the human protein Q9BU64 (accession numbers for protein and cDNA entry are tr: Q9BU64 and embl: BC 002870). In the database, the murine orthologue to this human protein 8430427C 03RIK or Q9BU64 is registered as protein Q9CXA4 (accession no. Tr: Q9CXA4). However, the investigations made within the scope of the invention have shown that the human sequence differs significantly from the murine sequence; it deviates strongly from the mouse sequence at the C-terminus of the protein and leads to a significantly longer ORF. In the human genome sequence (accession numbers embl: AC 013459 and embl: AC 012073) no counterpart to the shorter mouse-specific sequence was found. Based on the composition of mouse EST sequences, according to the invention it has been possible to obtain a mouse cDNA which, although deviating from the sequence stored in the database, clearly corresponds to the human sequence. Therefore, the in the database under the accession no. tr: Q9CXA4 stored sequence not correct. The amino acid or nucleic acid sequence obtained according to the invention is disclosed in US Pat 20 and 21 shown. In the present case, the term "BAB31214 / Q9BU64" is used for both the human protein and the mouse protein, unless a specific species is listed.

BAB31214 contains a coiled-coil region at the N-terminus, positions 1 to 110. It is believed that this region plays a role in the oligomerization of the protein and serves to interact with other coiled-coil proteins. Examination of the expression pattern in different tissues revealed an accumulation of mRNA only in the brain and in the spleen (see 22 ). Accordingly, a selective expression of BAB31214 in the nervous tissue can be assumed, so that BAB31214 is suitable for the development of low-side-effects analgesics. This is accomplished by cellular localization of the BAB31214 transcript in the gray matter of the spinal cord in motor neurons and in the brain.

OPA-1 / KIAA0567

These are the human protein OPA-1 / KIAA0567, which is designated in the database as O_060313: The rat and mouse orthologous proteins are registered in the database with NP_598513 and NP_598269, respectively. In the present case, the term "OPA-1 / KIAA0567" is used for both the human protein and the mouse and rat proteins unless specific species is listed. OPA-1 / KIAA0567 has a GTPase domain, a central dynamin domain and a high-basi N-terminal region responsible for mitochondrial localization (Delettre et al., 2000). It has been described by Olichon et al. (2003) assumed that OPA-1 / KIAA0567 as a component of mitochrontrial networks is an important organizer of the inner mitochondrial membrane, which is responsible for the integrity of the so-called Cristae. It has been found in the prior art that a mutation in the OPA-1 / KIAA0567 gene causes autosomal dominant hereditary optic atrophy type 1 (Alexander et al., 2000), which leads to blindness. It was therefore assumed that mutations in the OPA-1 / KIAA0567 gene disrupt the mitochondrial integrity and thus lead to reduced energy production in the nerve cells of the optic nerve. It is known that nerve cells are extremely sensitive to mitochondrial disorders due to their high energy requirements. However, there are also approaches in the prior art which question a purely mitochondrial localization due to a proven expression of the murine orthologue of OPA-1 / KIAA0567 in the brain (Misaka et al., 2002). According to the invention, OPA-1 / KIAA0567 was now isolated as a protein of synaptic membranes of the dorsal horn, whose expression could be detected mainly in the brain and for which ubiquitous expression, as is typical for mitochondrial proteins, could not be detected. These findings according to the invention and the fact that OPA-1 / KIAA0567 belongs to the mitochondrial proteins which are coded in the nuclear genome and thus can occur in other organelles, in contrast to the proteins coded in the mitochondrial genome, prove that OPA -1 / KIAA0567 is not a pure mitochondrial protein.

The studies of the expression pattern in various tissues on which the present invention was based revealed the strongest accumulation of the OPA-1 / KIAA0567 transcript in the brain (see 33 ), followed by the placenta and the uterus. In the other examined tissues (heart, lung, liver, kidney, spleen, stomach, small intestine, thymus, testes, skeletal muscle) no transcript was detected. A cellular localization of OPA-1 / KIAA0567 in pain-relevant tissue could be detected in the DRGs and in the gray matter of the spinal cord, in the motor neurons and in the brain (see p. 34 to 38 ). Based on the proven relatively selective expression of OPA-1 / KIAA0567 in the nervous system, it can be assumed that OPA-1 / KIAA0567 is suitable for the development of low-side-effects analgesics.

CPG2 / KIAA1756

at this protein is the cortical plasticity related protein 2 (CPG2), registered under accession number NP_062228. It is the rat orthologue to the human protein KIAA1756 (Accession Number BAB21847) and the murine protein NP_700448. In the present case, the Designation "CPG2 / KIAA 1756 "synonymous with the humane and mouse and rat proteins, unless specific Species listed The rat CPG2 protein was used as a protein of synaptic membranes isolated from the Dorsalhorn and has a length of 491 amino acids, contains nine Spectrin domains with about 100 amino acids in length and is similar to the Duchenne muscular dystrophy protein dystrophin with total 25 spectrin domains. Furthermore contains the CPG2 has an amino acid segment of 14 amino acids, of which 11 amino acids identical to a similar one long section of the dystrophin potein (Nedivi et al., 1996). It could be a cellular Localization of dystrophin in the brain as part of the postsynaptic density (PSD) (Lidov et al., 1990). PSD plays an important role in synaptic plasticity. Also CPG2 was associated with synaptic plasticity in the hippocampus and cortex brought. Both PSD and the synaptic plasticity of the dorsal horn neurons is involved in the chronification of pain. Next the fact known in the art, that with CPG2 structurally related protein dystrophin in the CNS is part of the PSD has been by the findings of the invention evidenced that CPG2 in both the dorsal horn of the spinal cord and in the PSD is expressed and thus at a central point of pain transmission is localized and to the development of low-side-effects analgesics suitable is.

LETM1

This is the human leucine zipper / EF-hand containing transmembrane protein 1 (LETM1) protein, which is designated NP_036450 in the database: The rat and mouse orthologous proteins are registered in the database with NP_062668 and XP_223541, respectively , In the present case, the term "LETM1" is used synonymously for the human protein as well as for the mouse and rat proteins, unless a specific species is listed. The human LETM1 gene could be localized by FISH analysis on chromosome 4p16.3. It is a protein 739 amino acids in length, contains two calcium-binding EF-hand domains, a DNA-binding leucine zipper motif, a transmembrane domain and several coiled-coil domains that mediate protein-protein interactions (Endele et al., 1999). It has also been found in the prior art that the human LETM1 gene is present in almost all wolf-staghorn patients Syndrome (WHS) (Endele et al., 1999), a hereditary disease characterized primarily by craniofacial dysmorphism, due to a chromosomal deletion missing at the end of the short arm of chromosome 4. This disease is associated with severe psychomotor retardation, often accompanied by hypotonia and cerebral seizures. The lack of human LETM1 is believed to be particularly responsible for the cerebral seizures of WHS patients (Zollino et al., 2003). Due to the finding according to the invention that LETM1 has been isolated as a protein of synaptic membranes of the dorsal horn in connection with the fact that human LETM1 plays a role in intracellular calcium homeostasis, whose intracellular modulation in turn represents an interesting target for spinal analgesia, proves that human LETM1 and LETM1 is generally a molecular target protein for the development of low-side-effects analgesics.

PID12832121 / FLJ20420

PID12832121 / FLJ20420 was isolated as a protein of synaptic membranes of the dorsal horn. It is the rat orthologue of the mouse protein 0610041L09RIK, the in the database with tr: Q9CRB9 and for the cDNA with NM 025336 listed is: The orthologous human protein is in the database with the accession numbers for protein and cDNA tr: Q9NX63 and embl: BC011596 registered. The rat orthologist is not registered in the database, but it could according to the invention a new Partial sequence of the cDNA obtained by composition of EST sequences become. In the present case, the term "PID12832121 / FLJ02420" is synonymous with the human protein and the Proteins from mouse and rat used unless special species listed will contain the mouse protein as well as the closely related relatives a (as yet unpublished) homology domain Mito-C4, from the MEMOREC Profile collection comes from (ME00694). This is in the amino acid sequence of the mouse domain in the area of amino acids 180 to 220. The Mito C4 domain is not associated with any special function so far. It deals a short homology domain of about 40 amino acids with four invariant cysteine residues found in many small proteins occurs. Most, if not all, of the proteins that make up the Mito C4 domain are localized in the mitochondrion, although they respectively a mitochondrial targeting sequence is missing. The domain exists from a 'helical hairpin 'unit, through two disulfide bridges is stabilized. This explains et al the four conserved Cys residues. Except this Mito-C4 domain finds no further homology domains in the PID12832121 / FLJ20420 protein. The Region of amino acid 50 to 180 contains a putative coiled-coil region which, as mentioned above, is a structural Motif represents that often the interaction with other coiled-coil proteins is used, in particular in the formation of homo-dimers. This coiled-coil area is only slightly conserved between PID12832121 / FLJ20420 and its closer relatives. The N-terminal region is more conserved, however, it did not resemble to proteins outside Found this subfamily. Furthermore, there are no signal or Transmembrane sequences. Due to the knowledge according to the invention, that PID12832121 / FLJ20420 as a protein of postsynaptic membranes of the Dorsalhorn was isolated that PID12832121 / FLJ20420 is suitable for the development of low-side-effects analgesics.

PGRL

in this connection it is the prostaglandin regulatory like protein PGRL. The orthologous PGRL proteins from rat, mouse and human are in the database for Protein and cDNA listed with the following accession numbers: Rat: XP_222900 (protein) and XM_222900 (cDNA), mouse: XP_110315 (protein) and XM_110315 (cDNA), human: NP_443100 (protein) and NM_052868 (CDNA). In the present case, the term "PGRL" becomes synonymous for the human protein as well as the mouse and rat proteins, unless specific species is listed. PGRL has one length of 613 amino acids and heard to the Ig superfamily. It has an N-terminal signal sequence, four immunoglobin domains, three N-linked Glycosylation sites, a transmembrane domain and a short cytoplasmic Range up. immunoprecipitation and mutational analyzes showed that PGRL with the tetraspanin protein CD81 interacts (Clark et al., 2001). The knowledge according to the invention, that PGRL transcripts strongest in the brain (followed by testis, heart and kidney and each further decreasing in liver, spleen, lung and skeletal muscle) showed that PGRL is involved in pain management and thus suitable for the development of low-side-effects analgesics.

According to the invention, a protein is also included and can be used in the method of the invention (method-protein), which is encoded by a nucleic acid which under stringent conditions with a nucleic acid sequence of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes. "Hybridization" in the sense of the invention means a base-paired formation of a double-stranded nucleic acid molecule from two separate single strands. "Stringente An "antisense" nucleic acid means a natural or modified nucleic acid, the base sequence of which is complementary to the base sequence of at least one subregion In other words, an "antisense nucleic acid" means the non-coding (antisense) strand of a nucleic acid which has the complementary base sequence to the coding (sense) strand of the nucleic acid.

Provided in the present specification, reference to the process proteins are also the functional fragments and functional Variants of proteins included. Furthermore, according to the invention, the terms "protein" and "polypeptide" are used interchangeably.

Also encompassed by the invention are functional fragments and functional Variants of the process protein or the defined below proteins according to the invention. "Functional" in the sense of the invention means that the proteins, fragments, variants play a role in pain in the organism in which they occur play, in particular at the pain regulation. This can e.g. an antinociceptive, antihyperalgesic or antiallodynic effect.

One fragment according to the invention refers to both proteins and polypeptides and nucleic acids of the present invention. This may be N-terminal, C-terminal or intrasequentially abbreviated amino acid or Nukleinsäuresquenzen act. The preparation of such fragments of the invention is in the state The technique is well known and can be applied by a professional performed by standard procedures (see, for example, Sambrook et al., 2001). In general, the Preparation of such fragments by modifying the nucleic acid sequence, which encodes the native protein or polypeptide, followed by a Transformation of this nucleic acid sequence into a suitable host and expression of this modified nucleic acid sequence, provided that the modification of the nucleic acid is the functional activities of the protein or polypeptide. Fragments of nucleic acids in the Meaning of the invention for example by treatment with suitable restriction enzymes getting produced. The identification of fragments according to the invention can either over the review of her Functionality through the measurement of their biological activity or by means of a sequencing of the fragments and a subsequent comparison the sequence obtained with the native sequence. Method of measuring the biological activity and sequencing methods are numerous in the art and well known.

variants The invention also relates to both the proteins to the polypeptides and the nucleic acids according to the present invention. As variants of proteins, polypeptides or nucleic acids or Fragments thereof, such proteins, polypeptides, nucleic acids or Fragments thereof, the sequence differences to the corresponding have native sequences. In these sequence deviations can it is one or more insertion (s), deletion (s) and / or Substitution (s) of amino acids act, with a sequence homology of at least 60%, preferably 70%, stronger preferably 80%, also stronger preferably 85%, even stronger preferably 90%, and most preferably 97%.

Around the percentage identity two nucleic acid or amino acid sequences to determine the Sequences are adjusted to the following matches or to determine deviations in the sequence. For this purpose, for. B. gaps into the sequence of the first amino acid or nucleic acid sequence introduced and the amino acids or nucleotides at the speaking position of the second amino acid or nucleic acid sequence be compared. If a position in the first amino acid sequence with the same amino acid or the same nucleotide is occupied, as it is at another Position in the second sequence is the case, then both sequences identical at this position. The percent identity between two sequences is a function of the number of identical positions divided by the sequences. The determination of the percentage identity of two Sequences can be performed using a mathematical algorithm. A preferred but not limiting example of a mathematical one Algorithm for the comparison of two sequences can be used is the Algorithm of Karlin et al. (1993), PNAS USA, 90: 5873-5877. One such algorithm is integrated in the NBLAST program with which sequences can be identified the one you want identity to the sequences of the present invention. To make a gap adjustment (also "gapped allignment ") like described above, the "Gapped BLAST" program can be used, as in Altschul et al, 1997, Nucleic Acids Res. 25: 3389-3402.

Under the term variants also fall in particular such amino acid and Nucleic acid sequences, the opposite the physiological sequences have conservative substitutions. Conservative substitution refers to such substitutions where amino acids be exchanged for each other, who come from the same class. In particular, there are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chain or amino acids whose Side chains of hydrogen bonds can be able to e.g. Side chains that have a hydroxy function. That means, that, for example, an amino acid having a polar side chain with another amino acid a likewise polar side chain is replaced or for example an amino acid characterized by a hydrophobic side chain another amino acid is substituted with also hydrophobic side chain, e.g. Serine (threonine), by threonine (serine) or leucine (isoleucine) by isoleucine (leucine). Insertions and substitutions are particular possible at such sequence positions, the no change the three-dimensional structure by insertion (s) or deletion (s) bring, which, for example, with the aid of CD spectra (circular dichroism spectra) easily verifiable.

Suitable methods for preparing variants which have substitutions with respect to the native sequences are described, for example, in the publications US 4,737,462 . US 4,588,585 and US 5,017,691 described. The preparation of variants in general is also described in particular by Sambrook et al., 2001. It codons can be omitted, supplemented or replaced. Variants may, in particular, also be those proteins, polypeptides or nucleic acids which are stabilized in order to counteract the physiological degradation, for example by stabilizing the protein backbone by substitution of the amide-like bond, for example also by the use of β-amino acids. Variants within the meaning of the invention can also be prepared in which changes are introduced into the nucleic acids which code the variants, such as, for example, insertions, deletions and / or substitutions of one or more nucleotides. Numerous methods for such alterations of nucleic acid sequences are known in the art. One of the most commonly used techniques is oligonucleotide-directed site-specific mutagenesis. In this technique, an oligonucleotide is synthesized whose sequence has a specific mutation. This oligonucleotide is then hybridized with a template containing the wild-type nucleic acid sequence. Preferably, a single-stranded template is used in this technique. After annealing of the oligonucleotide and template, a DNA-dependent DNA polymerase is used to synthesize the second strand of the oligonucleotide that is complementary to the template DNA strand. As a result, a heteroduplex molecule containing a mismatch resulting from the above-mentioned mutation in the oligonucleotide is obtained. The oligonucleotide DNA is introduced into a suitable plasmid, this is introduced into a (host) cell, in this (host) cell, the oligonucleotide DNA is replicated. This technique contains nucleic acid sequences with targeted alterations (mutations) which can be used for the preparation of variants according to the invention.

According to the invention is in Step (b) of the method according to the invention determines whether it is in the test substance used in the process is a pain-regulating substance. This can either be over the Binding of the test substance to the protein, for example through the repression a known ligand, or by determining the amount of bound Substance, or by determining the change of a functional Parameters as a result of the interaction between test substance and Protein done.

In a preferred embodiment Therefore, the measurement of the binding of a test substance to a Process protein over the repression a known labeled ligand of the protein (or functional Fragment or the functional variant thereof) and / or via the attached activity of a labeled test substance. Under "binding" of the test substance to the protein is to understand an interaction between protein and test substance, which leads to a fixation. Such an interaction can be found, for example, in the regulation, Inhibition and / or activation of receptors, ion channels and / or Enzymes take place. Under "displacement" of a known labeled ligand is a complete or partial removal to understand this ligand from its binding site. The ligand For example, it may be radioactive, fluorescent or luminescent be marked. Such a label makes the ligand corresponding Detection reactions (e.g., radioactivity, fluorescence or luminescence) accessible. A ligand is any substance that is highly specific to a molecule binds, which is found in the organism, especially on the surface of a Cell, located. An example of this is a receptor. This Ligand is bound by the also binding to such a molecule Test substance displaced from its binding site.

In a further preferred embodiment, the measurement of a parameter changed by the binding of the test substance to a process protein takes place. Under "functional parameters" are the measurement understanding the sizes of an experiment related to the function of the protein. Functional parameters can be, for example, the nexpression, the ion milieu, the pH, the membrane potential, the enzyme activity or the concentration of the 2 ^nd messengers. Preferably, at least one functional parameter changed by the test substance is measured by measuring the regulation, inhibition and / or activation of receptors, ion channels and / or enzymes, in particular by measuring the change in gene expression, ion milieu, pH, membrane potential, the enzyme activity or the concentration of the 2 ^nd messengers. Thus, according to the invention, the effect of the test substance can be directly affected by its influence on receptors, ion channels and / or enzymes, but it can also be done indirectly by measuring conditions such as gene expression, ionic milieu, pH, membrane potential, enzyme activity or concentration of the 2 ^nd messengers. "2 ^nd messenger" is understood to mean a small molecule which, as a consequence of an extracellular signal, is either formed in the cytosol or transported into the cytosol in order to forward information to the cell interior (eg cAMP, IP ₃ ) 2 ^nd messenger messengers of the intracellular signaling pathway are cyclic AMP (cAMP), inositol triphosphate (IP3) or diacylglycerol (DAG).

A Test substance is based on the results of those described above Measure measurements as pain-regulating when passing through their invasion into a living organism is a behavioral change causes the expert as analgesic (antinociceptive, antihyperalgetic or antiallodynic). In the method according to the invention For example, a substance can be called a pain-regulating substance if stronger Binding or triggering a change a functional parameter to a greater extent, (for example 100%), as compared to the average of the tested ones Substances take place.

In a preferred embodiment According to the invention, the cell synthesizing a process protein is preferably prior to step (a) of the method of the present invention manipulated. Below this is a change of cells, tissues or organisms understood in the genetic material in the cell In particular, this is genetic Material around one or more nucleic acids. It is preferred that the genetic manipulation involves the measurement of at least one changed a test substance functional parameter allowed. In this case, by the genetic manipulation a change a functional parameter at all or even improved. It is particularly preferred hereby that by genetic manipulation the cell is a non-endogenous expressed form of a G-protein (GTP-binding protein) expressed or that a reporter gene is inserted into the cell is therefore preferred a G protein is introduced into the cell that is either not endogenous is present or is not physiologically expressed. in this connection it may be, for example, a chimeric G protein, by that one change the signaling pathway, or a promiscuous G protein, which is a very binding protein. The term "G protein" is the internationally common abbreviation for a guanosine triphosphate (GTP) -binding protein. It is called signaling protein by G-protein activated coupled receptors. The term "reporter gene" is used for genes whose Products are characterized by simple biochemical or histochemical Just have methods proven. Examples are the Luciferase gene, the alkaline phosphatase or the green fluorescent protein (GFP). "Endogenous expresses "means that a cell or cell line under suitable culture conditions a protein expressed without any expression of this protein (by genetic manipulation) was induced. The introduction of a Reporter gene in a cell of the invention allows the measurement an induced expression of the gene product.

In a likewise preferred embodiment The invention relates to the genetically engineered cell prior to step (a) the process is cultured under conditions involving expression allow, preferably under selection pressure "cultivating" a cell means to keep the cell under conditions that will survive this cell as well ensure successor generation (s), i.e. especially under conditions that allow expression. Conditions that allow expression are from the protein dependent, that it is to be expressed. Such conditions relate, for example on the temperature, the pH, the medium used in which a cell is cultured, the Addition of inducing substances, nutrients and co-factors, Incubation time and oxygen content. A cultivation under "selection pressure" means that only selected Cells are further cultivated. This can be used to check whether the genetic engineering Manipulation has been successful. It is preferred for this purpose in cells that are genetically engineered, a particular gene introduced, its gene product, a so-called selection marker, only the cells a survival among the chosen ones Allows culture conditions, which contain this gene. An example of this is the introduction of a Antibiotic resistance gene.

A cell according to the present invention is preferably an amphibian cell, bacterial cell, yeast cell, insect cell or an imortalized or native mammalian cell. Examples of amphibian cells are Xenupus oocytes, for E. coli bacter cells, Bacillus, Pseudaomonas, Streptomyces, and Samonella, for yeast cells Saccharomyces cerevisiae, Pichia Pastoris, for insect cells Sf9 cells, for immortalized mammalian Hela cells and for mammalian native cells CHO cells and COS cells. However, this list is by no means exhaustive. The choice of a cell to be used in the method according to the invention depends on several factors, eg in the introduction of a vector into the cell, in particular of the vector used.

In a preferred embodiment, the cell is genetically engineered to contain at least one nucleic acid according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or a functional fragment or a functional variant thereof. In this way it can be achieved that a process protein is synthesized that is not endogenously expressed in a cell according to the invention. In this case, it is preferred that the nucleic acid is contained in a recombinant DNA construct, preferably a vector, particularly preferably an expression vector. A recombinant DNA construct is understood as meaning an in vitro produced DNA molecule. A vector is generally understood to mean a nucleic acid molecule which contains foreign genes (foreign nucleic acid).

at all Vectors mentioned in the present invention may be an expression vector, i. around a vector, the ability for expression and / or amplification of the nucleic acids contained in a prokaryotic and / or eukaryotic cell. Especially For example, the present invention relates to plasmid vectors, e.g. pBABEpuro, Phage or retroviral vectors, especially all vector systems, which may be used therapeutically, e.g. also adenoviral and adenoviral-associated vector systems.

Vectors of the invention preferably have control sequences which express the expression of enable nucleic acid according to the invention or reinforce and regulate transcription. Such control sequences include, for example. Polyadenylation signals, promoters, e.g. natural or synthetic promoters, Enhancer to effect transcription, operator sequences to Regulation of transcription, silencers for tissue-specific transcription, Sequences, the appropriate ribosome binding sites on the mRNA encode sequences that stabilize the mRNA and sequences, which regulate the termination of transcription and / or translation. This merely represents an exemplary listing of possible control sequences. Other possible Control sequences are well known in the art and are, for example,. described by Goeddel, 1990. The control sequences can be modified be, e.g. by deletion, addition or substitution of one or several nucleic acids, to strengthen their control function.

It In particular, there are many different promoters for different ones Organisms known. For example, a preferred vector promoter is used in Bacillus subtilis, the AprE promoter preferred vector used in E. coli is the T7 / Lac promoter, more preferred Promoter used in Saccharomyces cerevisiue is PGK1, a more preferred Promoter used in Aspergillus niger is glaA, a preferred one Promoter, used in Trichoderma reesei (reesei) is cbhI. Promoters the for the use in prokaryotic (host) cells are suitable, shut down e.g. beta-lactamase (vector pGX2907 [ATCC39344], contains the replicon and the beta-lactamase gene), lactose promoter systems (Chang et al., (1978) Nature (London, 275: 615); Goeddel et al. (1979), Nature (London), 281: 544), alkaline phosphatase, the tryptophan (trp) promoter system (Vector pATH1 [ATCC37695]) and hybrid promoters such as the tac promoter (isolable from plasmid pDR540 [ATCC37282]). But also other bacterial promoters whose nucleic acid sequences are well known enable a person skilled in the art to ligate it with a nucleic acid according to the invention, where also linker or adapter can be used to desired To create restriction sites. Preferably, promoters, which are used in bacterial systems, also a Shine-Dalgarno sequence, the functional with the nucleic acid connected is.

Suitable expression vectors may, for example, consist of segments of chromosomal, non-chromosomal and synthetic DNA. For this purpose, numerous derivatives of SV40 and bacterial plasmids are known. Examples are plasmids from E. coli such as col E1, pBK, pCR1, pBR322, pM69, pUC19 and their derivatives, plasmids useful for a broad host range, such as RP4, and phage DNAs, such as numerous derivatives of phage lambda , eg NM989, as well as other DNA phages, eg M13, as well as filamentous single-stranded DNA phages, yeast plasmids, vectors suitable for use in eukaryotic cells, as well as vectors consisting of a combination of plasmid and phage DNA. Numerous expression techniques for the use of expression vectors according to the invention are known in the prior art known. Such techniques are generally described, for example, in Sambrook et al., 2001.

Another object of the invention relates to a protein (referred to herein as "protein of the invention"), which has an amino acid sequence according to one of 11 . 20 or 54 or a functional fragment or a functional variant thereof, or consists of one of said amino acid sequences. As already described above, the amino acid sequence was determined according to 11 coding for human KIAA0378, completed by 149 amino acids compared to the amino acid sequence present in the database at the N-terminus. The amino acid sequence according to the 20 , coding for the murine BAB31214, as well as the amino acid sequence according to 54 , Rat coding for the protein PID12832121 / FLJ20420 from the rat were also obtained according to the invention. Also in this regard, reference is made to the above statements.

Another object of the invention is a protein (referred to herein as "protein according to the invention"), which by a nucleic acid according to one of the 12 . 21 or 55 A further subject matter of the invention is a protein which is encoded by a nucleic acid which can be amplified under stringent conditions with a nucleic acid according to one of the 12 . 21 or 55 or whose antisense nucleic acid hybridizes. The nucleic acid sequences of 12 . 21 and 55 encode human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420, respectively.

The invention also encompasses both the above-described proteins according to the invention (human KIAA0378, murine BAB31214 and rats PID12832121 / FLJ20420) and also the process proteins which have modifications. For example, the proteins may be posttranslationally modified (after translation), eg glycosylated, phosphorylated, amidated, methylated, acetylated, ADP-ribosylated, hydroxylated, membrane-anchored, cleaved or shortened. Post-translational modifications are described, for example, in Voet / Voet, Biochemistry, 1 ^st Edition, 1990, pp. 935-938.

The Production of process proteins and proteins according to the invention can be done by standard methods well known to those skilled in the art are. For example, such a method may typically be the comprising the steps of: (a) culturing a cell under suitable conditions Conditions, (b) expression of the nucleic acid encoding the protein or nucleic acid construct under appropriate conditions, and (c) isolating the protein the cells and / or the culture supernatant.

suitable Conditions for cultivating a cell have already been described above. The expression of the polypeptide can typically be up to date the technique in suitable expression systems, preferably as a secreted product of stable transfectants, e.g. CHO cells or other animal cells, such as Cos7 or SF9 (insect cells), or other eukaryotic cell systems, for example Pichia pastoris. Preferably, the expressed proteins of the invention respective leader sequences suitable for secretion in the cell system on. Therefore, vectors of the invention used for expression contain coding sections that are responsible for a functional leader sequence encode, e.g. as in Brocks et al. (Immunotechnology 3: 173-184, 1997) for mammal and insect cells or using, for example, pPICZalpha vectors (Invitrogen, Karlsruhe, Germany) for expression and secretion in the yeast Pichia pastoris. Isolating the protein of the invention from the (host) cell can be determined by standard methods, such as chromatography, precipitation etc. suitable for purification of polypeptides and proteins are made (see Sambrook et al., 2001).

One Another object of the method is a nucleic acid, the for a Protein consisting of the group consisting of human KIAA0378, murine BAB31214 and rats PID12832121 / FLJ20420 is selected, or a functional Fragment or a functional variant of such a protein, coded. Such a nucleic acid is referred to herein as "nucleic acid according to the invention".

Likewise an object of the present invention is a nucleic acid which consists of a nucleic acid sequence according to one of the 12 . 21 or 55 or comprises such a nucleic acid sequence or a functional fragment or a functional variant of such a nucleic acid. Such a nucleic acid is referred to herein as "nucleic acid according to the invention".

at all nucleic acids referred to in the present invention can It is both DNA, genomic DNA, synthetic DNA, in particular cDNA, as well as RNA, in particular mRNA act. The nucleic acid molecules can double or single-stranded available. single RNA or DNA can be either the coding (sense) or the non-coding (antisense) Be strand.

Also includes are nucleic acids, as nucleic acid constructs occur, so one for one of the proteins according to the invention coding sequence section and one or more further sequence section (s) contain. Such further sequence segments may be, for example, sequences be that for encode a leader peptide that acts as a signal for secretion of the protein according to the inventions in eukaryotic cells. Other sequences comprising the nucleic acids according to the invention could be, are also non-coding Sequences such as 3 'and 5' non-coding sequences including e.g. regulatory sequences.

The nucleic acid sequences of the present invention also with nucleic acid sequences be merged, for example, for encode a marker sequence or code for a sequence that a polypeptide encoding, for example, the isolation or purification of the polypeptide of the invention. Representative sequences include, for example. those containing a glutathione-S-transferase (GST) fusion protein Polyhistidine (e.g., HIS 6) hemagglutinin or encode HSV tag. However, this list is by no means restrictive The nucleic acids according to the invention can preferably isolated. This means that the nucleic acid molecule or the nucleic acid sequence not of nucleic acid sequences usually flanking the gene or the nucleic acid sequence flank (as in genomic sequences) and / or the complete or have been partially purified (such as in a DNA or RNA library). For example. can be an isolated nucleic acid of the Invention relating to the cellular environment in which it naturally Occurs, be isolated.

Also according to the invention functional fragments or functional variants of the nucleic acids according to the invention or nucleic acid constructs includes, to the above statements to the terms functional, fragment and variant corresponding Find application.

The Production of Nucleic Acids According to the Invention, Nucleic Acid Constructs or functional fragments or functional variants thereof can be carried out by standard methods known to those skilled in the art (see, e.g., Sambrook et al., 2001). In particular, application In this context, the PCR technique is a review of the Sequence of produced nucleic acids according to the invention can by means of sequencing or hybridization methods, which the expert also familiar are.

Also encompassed by the invention are gene products of the nucleic acids according to the invention. Preferably, the gene product encodes a protein or polypeptide in the 12 . 21 or 55 represented amino sequences. A gene product of the present invention concerns not only the transcript (mRNA) but also polypeptides or proteins, preferably in purified form. Also included are alleles, functional fragments or functional variants of such gene products. For functional fragments or functional variants of the gene products, the above definitions of these terms apply accordingly.

• – eine Nukleinsäure enthält, die für ein Protein, ausgewählt aus der Gruppe bestehend aus humanes KIAA0378, murines BAB31214 und Ratten PID12832121/FLJ20420, oder ein funktionelles Fragment oder eine funktionelle Variante eines solchen Proteins codiert und/oder
• – eine Nukleinsäure enthält, die aus einer Nukleinsäuresequenz gemäß einer der 12, 21 oder 55 besteht, oder eine solche Nukleinsäuresequenz umfasst oder ein funktionelles Fragment oder eine funktionelle Variante einer solchen Nukleinsäure und/oder
• – eine Antisense Nukleinsäure oder eine PNA enthält, die eine Nukleinsäuresequenz aufweist, die in der Lage ist, spezifisch an eine Nukleinsäure zu binden, die eines der Proteine humanes KIAA0378, murines BAB31214 und Ratten PID12832121/FLJ20420 codiert bzw. spezifisch an eine Nukleinsäure gemäß der 12, 21 oder 55 bindet.

Another object of the invention is a vector that at least

• A nucleic acid coding for a protein selected from the group consisting of human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420, or a functional fragment or a functional variant of such a protein and / or
• Contains a nucleic acid which consists of a nucleic acid sequence according to one of the 12 . 21 or 55 or comprises such a nucleic acid sequence or a functional fragment or a functional variant of such a nucleic acid and / or
• Contains an antisense nucleic acid or a PNA which has a nucleic acid sequence capable of binding specifically to a nucleic acid which encodes one of the proteins human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420 or specifically to a nucleic acid according to the 12 . 21 or 55 binds.

at such a vector according to the invention it is preferably one of the vectors described above.

• – ein Protein enthält, das eine Aminosäuresequenz gemäß einer der 11, 20 oder 54 oder ein funktionelles Fragment oder eine funktionelle Variante hiervon umfasst oder aus einer der genannten Aminosäuresequenzen besteht und/oder
• – ein Protein enthält, das durch eine Nukleinsäure gemäß einer der 12, 21 oder 55 codiert wird und/oder
• – ein Protein enthält, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure gemäß einer der 12, 21 oder 55 oder deren Antisense Nukleinsäure hybridisiert und/oder
• – eine Nukleinsäure enthält, die für ein Protein, ausgewählt aus der Gruppe bestehend aus humanes KIAA0378, murines BAB31214 und Ratten PID12832121/FLJ20420, oder ein funktionelles Fragment oder eine funktionelle Variante eines solchen Proteins codiert und/oder
• – eine Nukleinsäure enthält, die aus einer Nukleinsäuresequenz gemäß einer der 12, 21 oder 55 besteht, oder eine solche Nukleinsäuresequenz umfasst oder ein funktionelles Fragment oder eine funktionelle Variante einer solchen Nukleinsäure und/oder
• – eine Antisense Nukleinsäure oder eine PNA enthält, die eine Nukleinsäuresequenz aufweist, die in der Lage ist, spezifisch an eine Nukleinsäure zu binden, die eines der Proteine humanes KIAA0378, murines BAB31214 und Ratten PID12832121/FLJ20420 codiert bzw. spezifisch an eine Nukleinsäure gemäß der 12, 21 oder 55 bindet und/oder
• – einen Vektor enthält, der eine der vorgenannten Nukleinsäuren enthält.

Another object of the invention is a cell that at least

• Contains a protein which has an amino acid sequence according to one of the 11 . 20 or 54 or comprises a functional fragment or a functional variant thereof or consists of one of said amino acid sequences and / or
• Contains a protein which is protected by a nucleic acid according to one of the 12 . 21 or 55 is coded and / or
• Contains a protein which is encoded by a nucleic acid which under stringent conditions with a Nucleic acid according to one of the 12 . 21 or 55 or whose antisense nucleic acid hybridizes and / or
• A nucleic acid coding for a protein selected from the group consisting of human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420, or a functional fragment or a functional variant of such a protein and / or
• Contains a nucleic acid which consists of a nucleic acid sequence according to one of the 12 . 21 or 55 or comprises such a nucleic acid sequence or a functional fragment or a functional variant of such a nucleic acid and / or
• Contains an antisense nucleic acid or a PNA which has a nucleic acid sequence capable of binding specifically to a nucleic acid which encodes one of the proteins human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420 or specifically to a nucleic acid according to the 12 . 21 or 55 binds and / or
• - Contains a vector containing one of the aforementioned nucleic acids.

Preferably If such a cell according to the invention is an amphibian cell, bacterial cell, Yeast cell, insect cell or immortalized or native mammalian cell. examples for such cells have already been described above.

The Production of cells according to the invention can be determined by methods well known in the art are (for example described in Sambrook, 2001) done. Such a method may comprise the following steps: (a) Preparation of an Inventive Nucleic Acid According to the Invention or vector of the invention, (b) introducing the nucleic acid and / or the vector according to step (a) in a cell. The manufacture of one or more described above nucleic acid or vector of the invention can be well known to those skilled in the art and after the above done standard procedures. The introduction of the nucleic acid or of the invention vector of the invention into the cell can be made using any suitable standard method respectively. These include for example, transformation, electroporation, transfection using from e.g. Calcium chloride, lipofection, infection, transduction etc. Various standard methods are described, for example, in Sambrook et al., 2001.

One Another object of the invention is an antibody to one of the proteins of the invention - human KIAA0378, murine BAB31214 and rats PID12832121 / FLJ20420. Under antibody the expert understands soluble or molecules bound to cell membranes by their specific Interaction with a complementary cell surface molecule ("antigen") characterized are. They are generally referred to as immunoglobulins Proteins that have a specific binding site for antigens. Preferably it is a monoclonal or polyclonal antibody. monoclonal antibody are selective against a single antigenic determinant of an antigen directed antibodies, while polyclonal antibody a mixture of antibodies represent directed against several determinants of an antigen are.

One Another object of the invention is an antisense nucleic acid, the a nucleic acid sequence comprises or from a nucleic acid sequence which is capable of specific to a nucleic acid according to the invention or a functional fragment or a functional variant of a to bind nucleic acid according to the invention.

Under an antisense nucleic acid is a nucleic acid to understand the complementary to a coding (sense) nucleic acid. An antisense nucleic acid can present as DNA or RNA. An antisense nucleic acid can for example so-called antisense strategies used by which the mRNA or protein concentration influenced, in particular reduced, can be. For example, derived from the nucleic acid sequence the complete cDNA or substructures thereof constructs are created, e.g. Antisense oligonucleotides (DNA or RNA), for example using modified nucleotide building blocks (e.g., O-allyl ribose) an increased stability across from Have nucleases. These modified antisense oligonucleotides can then, for example, bind to a complementary mRNA and so their Prevent or hinder translation. Furthermore, this antisense constructs are useful, those using non-traditional bases such as inosine, quinine or Wybutosine as well as acetyl, methyl, thio and similarly modified Forms of adenine, cytidine, guanosine u, thymidine and uridine do not or degraded to a lesser extent by endogenous nucleases can. An antisense nucleic acid The present invention preferably has a length of 15 to 100 nucleotides, stronger preferably from 15 to 50 nucleotides, more preferably from 15 to 30 Nucleotides on strongest preferably 18 to 25 nucleotides.

One Another object of the invention is a PNA containing a nucleic acid sequence comprises or from a nucleic acid sequence which is capable of specific to a nucleic acid according to the invention or a functional fragment or a functional variant of a to bind nucleic acid according to the invention. PNA represents the internationally common abbreviation for Peptidic Nucleic Acid ("peptidic nucleic acid") Nucleic acid "). Form this linked pepditisch amino acids a chain, where the amino acids as a side chain one for have hybridization with a base suitable for DNA or RNA. PNAs can by replacing the phosphate backbone in nucleic acid sequences getting produced. A PNA according to the present Invention preferably has a length of 15 to 100 nucleotides, stronger preferably from 15 to 50 nucleotides, most preferably from 15 to 30 nucleotides on.

Another object of the invention is a ribozyme comprising a nucleic acid sequence which is capable of binding specifically to a nucleic acid according to the invention or a functional fragment or a functional variant of a nucleic acid according to the invention. Likewise, a nucleic acid according to the invention may be part of a ribozyme or another DNA enzyme or a catalytic RNA or DNA. A ribozyme is understood as meaning a catalytically active ribonucleic acid which, as an enzymatically active RNA molecule, has a specific cleavage of the RNA value. These include self-splicing RNAs. Examples of ribozymes are, for example, self-splicing introns, ribonuclease P (from E. coli), the hammerhead ribozyme, the hairpin ribozyme or tRNA ^Phe . Soche ribozymes may contain sections of nucleic acids that bind to and cleave complementary nucleic acids (sections of nucleic acid). For example, the function of self-splicing RNA is to remove introns from pre-rRNAs and pre-tRNAs, leading to mature rRNAs or tRNAs. The splicing reaction is initiated by the contact of a G nucleotide with the intron of the RNA. The RNA is cleaved, the new 3 'end of the RNA generated by this cleavage, in turn, cleaves the other end of the intron. A ribozyme according to the present invention preferably has a length of from 20 to 100 nucleotides, more preferably from 40 to 90 nucleotides, most preferably from 50 to 70 nucleotides.

One Another object of the invention is an siRNA which has a nucleic acid sequence comprises or from a nucleic acid sequence which is capable of specific to a nucleic acid according to the invention or a functional fragment or a functional variant of a to bind nucleic acid according to the invention. siRNA stands for "short interference RNA ". It is an intermediate of the "RNA interference" (RNAi) signaling pathway This is a double-stranded RNA molecule through Dicer ribonuclease into small interfering RNAs (siRNAs) broken up. One of the strands the siRNA, namely the strand complementary to a target mRNA, binds to this mRNA, which then degrades by the RDE-1 nuclease becomes. An siRNA according to the present Invention preferably has a length of 10 to 50 nucleotides, more preferred from 15 to 40 nucleotides, stronger preferably from 20 to 25 nucleotides. And most preferred 21 to 23 nucleotides. About that In addition, so-called sh (small hairpin) RNAs provide another Subject of the invention in the context of the present invention they are preferably double-length RNA sequences, such as above for described siRNA, which also bind to a nucleic acid according to the invention can. Such shRNAs are preferably expressed by transcription a plasmid, wherein the finished RNA transcript by refolding a double-stranded one RNA can form with a hairpin loop. Preferably, therefore Such a shRNA (a special form of siRNA) one to a the nucleic acid strands of nucleic acid sequence complementary to the invention which as such form a double strand with a hairpin loop can.

such shRNA have a slowed dissociation kinetics

Preferably, siRNAs of the invention have the general structure 5 '- (N _19-25 ) -3', more strongly 5 '- (N _19-24 ) -3', even more preferably 5 '- (N _21-23 ) -3' where N is any base. In this case, at least 90%, preferably 99% and in particular 100% of the nucleotides of a dsRNA according to the invention can be complementary to a section of the (m) RNA sequence of a sequence according to the invention. 90% complementary here means that, for example, given a length of 20 nucleotides of an siRNA according to the invention, this is not complementary to the corresponding segment on the (m) RNA for at most 2 nucleotides. The sequence of the double-stranded RNA according to the invention, with its general structure, is preferably completely complementary to a section of the (m) RNA of a sequence according to the invention.

Further preferred are siRNA _{according to the} invention which have the following sequence motifs: AAN _I9 TT, NAN ₁₉ NIN, NARN ₁₇ YNN and / or NANN ₁₇ YNN, where N is an arbitrary nucleotide, A is adenosine, T is thymidine, R is purines (A or G) and Y is pyrimidine bases (C or T).

In principle, a dsRNA according to the invention can be added to any desired section on the mRNA or the primary transcript will be complementary to a sequence according to the invention.

In A eukaryotic cell is used for the production of an mRNA the gene in its entire length, both introns and exons, transcribed into a long RNA molecule, the primary transcript. The stability of mRNA is made by processing the primary transcript at the 5 'end with an addition of an untypical nucleotide with a methylated guanine and a Polyadenylation at the 3 'end. Before the RNA leaves the nucleus, be through RNA splicing removed the intron sequences and linked the exons together, especially opposite the splice form, that is, the mature mRNA, an siRNA according to the invention is complementary.

A particularly preferred embodiment is a sh / siRNA that has a GC content of at least 30%, in one stronger preferred embodiment from to 30% to 70%, and in a more preferred embodiment from 40% to 60% or even stronger preferably between 45 and 55%.

A Another particularly preferred embodiment of an inventive si (sh) RNA is a target sequence that has the same frequency of all nucleotides contains the antisense strand. After all it is particularly preferred for 2'-deoxythymidine to be the 2-nt 3'-overhang occurs in an siRNA according to the invention, since this is thereby protected from exonuclease activity. Further preferred it is when the target sequence of a si / shRNA according to the invention only once in the target genes occurs or singular for the respective genome of the treated Cells is.

Preferably can the ends of the double-stranded ones RNA (siRNA o. ShRNA) can be modified to undergo degradation in the cell or to counteract a dissociation into the single strands, in particular to avoid premature degradation by nucleases.

A regularly not desired Dissociation of single strands of si / shRNA occurs especially when using low concentrations or short chain lengths on. For particularly effective inhibition of dissociation by the the nucleotide pairs brought cohesion of the double-stranded structure of siRNA according to the invention by at least one, preferably more chemical linkage (s) increase. An siRNA according to the invention, their dissociation is reduced, has a higher stability against enzymatic and chemical degradation in the cell or organism or ex-vivo on.

The chemical linkage the single strands an siRNA according to the invention is expediently by a covalent or ionic bond, hydrogen bonding, hydrophobic interaction, preferably van der Waals or stacking interactions, or formed by metal ion coordination. She can after one particularly advantageous embodiment feature on at least one, preferably at both ends. It has continued proved to be advantageous that the chemical linkage means one or more connection groups is formed, wherein the Compound groups preferably poly (oxyphosphinicooxy-1,3-propanediol) - and / or polyethylene glycol chains are The chemical linkage can also by in the double-stranded Structure instead of purines used purine analogues are formed. It is also advantageous that the chemical linkage by in the double-stranded structure introduced Azabenzene units can also be formed by in the double-stranded structure formed in place of nucleotides used branched nucleotide analogues become.

It has proved to be useful at least one of the following to produce the chemical linkage Groups used is methyl blue; bifunctional groups, preferably Bis (2-chloroethyl) -amine; N-acetyl-N '- (p-glyoxybenzoyl) -cystamine; 4-thiouracil; Psoralen. Furthermore, the chemical linkage can be carried by at the ends of the ds Area attached thiophosphoryl groups are formed. Preferably becomes the chemical linkage at the ends of the double-stranded Area by triple helix bonds produced. The chemical linkage may suitably be induced by ultraviolet light.

The Modification of the nucleotides of the dsRNA leads to deactivation one at double-stranded RNA dependent Protein kinase (PKR), in the cell. The PKR induces apoptosis. advantageously, is at least one 2'-hydroxy group the nucleotides of the siRNA in the double-stranded structure by a chemical group, preferably a 2'-amino or a 2'-methyl group replaced. At least one Nucleotide in at least one strand of the double-stranded structure can also be a so-called "locked nucleotide "with one, preferably by a 2'-O, 4'-C-methylene bridge, chemical be modified sugar ring. Advantageously, several Nucleotides "locked nucleotides ".

modifications the nucleotides of si / shRNA according to the invention concerns especially the dissociation of the nucleotides by amplification of the nucleotides Hydrogen bonding. The stability the nucleotide is increased and protected against attack by RNAses.

Furthermore can be done by modifying the backbone from the si / shRNA according to the invention prevent premature degradation. Especially preferred is dsRNA encoding (phosphorothioate, 2'-O-methyl RNA, LNA, LNA / DNA gapmer) is modified and therefore a longer one Having half-life in vivo.

The si / shRNA is prepared by methods known to those skilled in the art, this nucleotides, especially oligonucleotides, for example in the manner of the Merryfield synthesis, on an insoluble support (H.G. Gassen, Chemical and Enzymatic Synthesis of Gene Fragments (publ Chemie, Weinheim 1982)) or otherwise synthesized (Beyer / Walter, Textbook of Organic Chemistry, 20th Edition, (S. Hirzel Verlag, Stuttgart 1984), p. 816 ff). The recovery of VGLUT mRNA can be achieved by Hybridization by genome and cDNA databases can be achieved. Si / shRNA molecules according to the invention can, for example, be. be prepared synthetically various providers, for example IBA GmbH (Göttingen, Germany) become.

Double-stranded RNA according to the invention, especially si / shRNA, can be included in micellar structures be the separation of substance groups in vitro and in vivo influence. In this case, the si / shRNA is preferably in liposomes in front. The liposomes are artificial, Globular self-contained membranes, phospholipids, in the encapsulating both hydrophilic substances in the aqueous interior, as well as lipophilic substances in the nenbereich of the lipid membrane can be incorporated. The requirement for the use of liposomes for experimental or therapeutic purposes is their compatibility with cells and tissues. The siRNA, preferably in the liposomes can be present with a peptide sequence, preferably with a lysine and arginine-rich sequence, for example, a sequence from the viral TAT protein (for example containing AS 49-57), to be used as transporter peptide to overcome the cell membrane more easily.

One Another object of the invention is an aptamer which has a nucleic acid sequence comprises or from a nucleic acid sequence which is capable of specific to a nucleic acid according to the invention or a functional fragment or a functional variant of a to bind nucleic acid according to the invention. Under an aptamer are nucleic acids or nucleic fragment fragments to understand protein-binding properties. This includes so-called Spiegelmers, the mirror image obtained by mirror evolution and thus represent stable oligonucleotides that are highly affine and highly specific a target molecule can bind (Klußmann et al., 1996). An aptamer according to the present Invention preferably has a length of 20 to 100 nucleotides, stronger preferably from 18 to 80 nucleotides, most preferably from 60 to 80 nucleotides.

• – ein Protein enthält, das eine Aminosäuresequenz gemäß einer der 11, 20 oder 54 oder ein funktionelles Fragment oder eine funktionelle Variante hiervon umfasst oder aus einer der genannten Aminosäuresequenzen besteht und/oder
• – ein Protein enthält, das durch eine Nukleinsäure gemäß einer der 12, 21 oder 55 codiert wird und/oder
• – ein Protein enthält, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure gemäß einer der 12, 21 oder 55 oder deren Antisense Nukleinsäure hybridisiert und/oder
• – eine Nukleinsäure enthält, die für ein Protein, ausgewählt aus der Gruppe bestehend aus humanes KIAA0378, murines BAB31214 und Ratten PID12832121/FLJ20420, oder ein funktionelles Fragment oder eine funktionelle Variante eines solchen Proteins codiert und/oder
• – eine Nukleinsäure enthält, die aus einer Nukleinsäuresequenz gemäß einer der 12, 21 oder 55 besteht, oder eine solche Nukleinsäuresequenz umfasst oder ein funktionelles Fragment oder eine funktionelle Variante einer solchen Nukleinsäure und/oder
• – eine Antisense Nukleinsäure oder eine PNA enthält, die eine Nukleinsäuresequenz aufweist, die in der Lage ist, spezifisch an eine Nukleinsäure zu binden, die eines der Proteine humanes KIAA0378, murines BAB31214 und Ratten PID12832121/FLJ20420 codiert bzw. spezifisch an eine Nukleinsäure gemäß der 12, 21 oder 55 bindet und/oder
• – einen Vektor enthält, der eine der vorgenannten Nukleinsäuren enthält und/oder
• – eine Zelle enthält, die eines/eine der vorgenannten Proteine, Nukleinsäuren und/oder den vorgenannten Vektor enthält.

Another object of the invention is a transgenic non-human mammal, the at least

• Contains a protein which has an amino acid sequence according to one of the 11 . 20 or 54 or comprises a functional fragment or a functional variant thereof or consists of one of said amino acid sequences and / or
• Contains a protein which is protected by a nucleic acid according to one of the 12 . 21 or 55 is coded and / or
• Contains a protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid according to one of the 12 . 21 or 55 or whose antisense nucleic acid hybridizes and / or
• A nucleic acid coding for a protein selected from the group consisting of human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420, or a functional fragment or a functional variant of such a protein and / or
• Contains a nucleic acid which consists of a nucleic acid sequence according to one of the 12 . 21 or 55 or comprises such a nucleic acid sequence or a functional fragment or a functional variant of such a nucleic acid and / or
• Contains an antisense nucleic acid or a PNA which has a nucleic acid sequence capable of binding specifically to a nucleic acid which encodes one of the proteins human KIAA0378, murine BAB31214 and rat PID12832121 / FLJ20420 or specifically to a nucleic acid according to the 12 . 21 or 55 binds and / or
• Contains a vector containing one of the aforementioned nucleic acids and / or
• - Contains a cell containing one of the aforementioned proteins, nucleic acids and / or the aforementioned vector.

In a preferred embodiment contain germinal and somatic cells of the transgenic nonhuman mammal one of the above-described nucleic acids by chromosomal introduction into the genome of the animal or into the genome of one of the ancestors of the said animal. Method for introducing nucleic acids into the genome of an animal is well known to those skilled in the art.

In a further preferred embodiment contain germinal and somatic cells of the transgenic nonhuman mammal one of the above-described nucleic acids by chromosomal manipulation of the genome of the animal or genome of one of the ancestors of the said Animal contained in no longer expressible form. Such chromosomal manipulation that prevents the genetic information (Nucleic acid) although it is present in the genome, it can not be transcribed for example, by regulatory sequences in addition to contain the coding region of the nucleic acid causes become. Likewise coding portions of the nucleic acid are removed, preferably by Cutting the nucleic acid by means of suitable restriction enzymes. Suitable restriction enzymes for this purpose can by the skilled person from the restriction sites of these enzymes in conjunction with the nucleic acid sequence be easily determined.

It it is particularly preferred that the transgenic non-human mammal is a rodent, especially a rat or a mouse.

One Another object of the invention is a compound by the inventive method is identifiable. Preferably, the compound is as a pain-regulating Substance identifiable. This is particularly preferred to a substance or a pain-regulating substance, as they already are has been described above. "Identifiable" means in this Related that the result of measuring the binding in step (B) of the method according to the invention a much stronger, preferably twice as strong, binding of the compound to the protein has as the average of the substances to be tested, or that the result of the change a functional parameter in the nature of the functional parameter or in the extent of change significantly different from the average of the substances to be tested.

In a further embodiment the invention, the proteins or polypeptides, but possibly also nucleic acids, Nucleic acid constructs, Vectors or Cells of the Invention (hereinafter referred to as "molecules of the invention") as a medicament or for the manufacture of a medicament for the treatment of diseases or as a diagnostic.

• (a) mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL und/oder mindestens ein Protein umfassend eine Aminosäuresequenz gemäß einer der 11, 18, 20, 27, 29, 31, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 und/oder mindestens ein Protein, für das eine Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 codiert, und/oder mindestens ein Protein, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 oder deren Antisense Nukleinsäure hybridisiert oder ein funktionelles Fragment oder eine funktionelle Variante eines der vorgenannten Proteine,
• (b) mindestens eine Nukleinsäure codierend für mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL oder ein funktionelles Fragment oder eine funktionelle Variante hiervon und/oder mindestens eine Nukleinsäure umfassend oder bestehend aus eine(r) Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 und/oder mindestens eine Nukleinsäure, insbesondere eine Antisense Nukleinsäure oder eine PNA, welche eine Sequenz aufweist, die in der Lage ist, spezifisch an eine der vorgenannten Nukleinsäuren zu binden und/oder ein funktionelles Fragment oder eine funktionelle Variante der mindestens einen Nukleinsäure,
• (c) mindestens einen Vektor enthaltend eine Nukleinsäure gemäß (b),
• (d) mindestens einen Antikörper gegen eines der Proteine oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a),
• (e) mindestens eine Zelle enthaltend mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a), mindestens eine Nukleinsäure gemäß (b), mindestens einen Vektor gemäß Punkt (c) oder mindestens einen Antikörper gemäß (d)
• (f) mindestens eine Verbindung gemäß einem der Ansprüche 25 oder 26 und/oder
• (g) mindestens einen Wirkstoff, der an mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a) bindet,

sowie gegebenenfalls geeignete Hilfs- und/oder Zusatzstoffe.Another object of the invention is therefore a medicament, the

• (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 encoded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the abovementioned proteins,
• (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid,
• (c) at least one vector containing a nucleic acid according to (b),
• (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a),
• (e) at least one cell containing at least one protein or a functional fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (d)
• (f) at least one compound according to one of claims 25 or 26 and / or
• (g) at least one active substance which binds to at least one protein or a functional fragment or a functional variant thereof according to (a),

and optionally suitable auxiliaries and / or additives.

Also according to the invention a combination of molecules of the invention with pharmaceutical suitable vehicle, Auxiliary and / or additives disclosed. Appropriate production routes are at "Remington's Pharmaceutical Sciences "(Mack Pub. Co., Easton, PA, 1980), which is part of the disclosure of present invention. For the parenteral administration of the medicaments and combinations according to the invention come as carriers For example, sterile water, sterile saline solutions, polyalkylene glycols, hydrogenated Naphthalene and in particular biocompatible lactide polymers, lactide / glycolide copolymer or polyoxyethylene / polyoxypropylene copolymers. Furthermore can medicaments of the invention filling substances or substances such as lactose, mannitol, covalent substances linking of polymers, e.g. Polyethylene glycol to inhibitors of the invention, Complexation with metal ions or inclusion of materials in or on special preparations of polymer compound, e.g. Polylactate, polyglycolic acid, hydrogel or on liposomes, microemulsion, micelles, unilamelar or multilamelar Vesicles, erythrocyte fragments or spheroplasts. Also may be included Solvent, diluent, Dyes and / or binders. The respective embodiments the medicines are dependent physical behavior, for example, in terms of Solubility, stability, bioavailability or degradability, to choose. Controlled or constant release of the active ingredient component according to the invention in the composition closes Formulations based on lipophilic depots (e.g., fatty acids, waxes or oils). In the context of the present invention, coatings of molecules according to the invention or Medicaments containing molecules according to the invention, discloses, in particular coatings with polymers, e.g. poloxamers or poloxamines. Furthermore you can molecules of the invention or inventive drugs protective coatings, e.g. Protease inhibitors or permeability enhancers.

The Choice of suitable carrier, Auxiliary and / or additives and the determination of the administered Dose hangs thereof from the route of administration of the drug. An inventive drug can be considered liquid Dosage forms in the form of injectable solutions, drops or juices, as semi-solid dosage forms in the form of granules, tablets, pellets, Patches, capsules, patches or aerosols (e.g., spray) become. in principle are used in the context of the present invention for medicaments according to the invention discloses all routes of administration known in the art, e.g. parenteral, oral, peroral, intravenous, intraperitoneal, intradermal, intramuscularly, intranasal, buccal, rectal or local, For example, on infections of the skin, mucous membranes and in the eyes. Preferably, the administration of a medicament according to the invention takes place parenteral, i. for example, subcutaneously, intramuscularly or intravenously, or orally or intranasal. For suitable for parenteral, topical or inhalative administration solutions, Suspensions, easily reconstitutable dry preparations as well Sprays. For oral administration is to preparations in the form of tablets, Dragees, capsules, granules, drops, juices and syrups. For a percutaneous Administered are the molecules of the invention in a depot in dissolved form or in a patch, optionally with the addition of the skin penetration färdernden Means, before. By oral or percutaneous administration, the Delay release of the molecules of the invention. The dose to be administered to the patient is of several factors dependent, for example, the weight of the patient, the type of application, the indication and the degree of the disease. Usually 2 to 500 mg / kg administered at least one molecule of the invention. If the drug is to be used for gene therapy, then For example, physiological saline, stabilizers, proteinase and / or DNAse inhibitors suitable auxiliaries and / or additives.

• (a) mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL und/oder mindestens ein Protein umfassend eine Aminosäuresequenz gemäß einer der 11, 18, 20, 27, 29, 31, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 und/oder mindestens ein Protein, für das eine Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 codiert, und/oder mindestens ein Protein, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 oder deren Antisense Nukleinsäure hybridisiert oder ein funktionelles Fragment oder eine funktionelle Variante eines der vorgenannten Proteine,
• (b) mindestens eine Nukleinsäure codierend für mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL oder ein funktionelles Fragment oder eine funktionelle Variante hiervon und/oder mindestens eine Nukleinsäure umfassend oder bestehend aus eine(r) Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 und/oder mindestens eine Nukleinsäure, insbesondere eine Antisense Nukleinsäure oder eine PNA, welche eine Sequenz aufweist, die in der Lage ist, spezifisch an eine der vorgenannten Nukleinsäuren zu binden und/oder ein funktionelles Fragment oder eine funktionelle Variante der mindestens einen Nukleinsäure,
• (c) mindestens einen Vektor enthaltend eine Nukleinsäure gemäß (b),
• (d) mindestens einen Antikörper gegen eines der Proteine oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a),
• (e) mindestens eine Zelle enthaltend mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a), mindestens eine Nukleinsäure gemäß (b), mindestens einen Vektor gemäß Punkt (c) oder mindestens einen Antikörper gemäß (d)
• (f) mindestens eine Verbindung gemäß einem der Ansprüche 25 oder 26 und/oder
• (g) mindestens einen Wirkstoff, der an mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a) bindet,

sowie gegebenenfalls geeignete Zusatzstoffe.Another object of the invention is a diagnostic that

• (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 encoded, and / or at least one protein which is encoded by a nucleic acid, which under stringent conditions with a nucleic acid to comprising a nucleic acid sequence according to any one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the abovementioned proteins,
• (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid,
• (c) at least one vector containing a nucleic acid according to (b),
• (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a),
• (e) at least one cell containing at least one protein or a functional fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (d)
• (f) at least one compound according to one of claims 25 or 26 and / or
• (g) at least one active substance which binds to at least one protein or a functional fragment or a functional variant thereof according to (a),

and optionally suitable additives.

A "diagnostic" within the meaning of the invention provides a tool for diagnosis, for example of a disease, A diagnostic agent which contains a nucleic acid is preferred which is an antisense nucleic acid or a PNA. Molecules of the invention may also be used used for in vitro diagnosis.

• (a) mindestens eines Proteins ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL und/oder mindestens eines Proteins umfassend eine Aminosäuresequenz gemäß einer der 11, 18, 20, 27, 29, 31, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 und/oder mindestens eines Proteins, für das eine Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 codiert, und/oder mindestens eines Proteins, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure umfassend eine Nuk leinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 oder deren Antisense Nukleinsäure hybridisiert oder eines funktionellen Fragments oder einer funktionellen Variante eines der vorgenannten Proteine,
• (b) mindestens eine Nukleinsäure codierend für mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL oder ein funktionelles Fragment oder eine funktionelle Variante hiervon und/oder mindestens eine Nukleinsäure umfassend oder bestehend aus eine(r) Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 und/oder mindestens eine Nukleinsäure, insbesondere eine Antisense Nukleinsäure oder eine PNA, welche eine Sequenz aufweist, die in der Lage ist, spezifisch an eine der vorgenannten Nukleinsäuren zu binden und/oder ein funktionelles Fragment oder eine funktionelle Variante der mindestens einen Nukleinsäure,
• (c) mindestens eines Vektors enthaltend eine Nukleinsäure gemäß (b),
• (d) mindestens eines Antikörpers gegen eines der Proteine oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a),
• (e) mindestens einer Zelle enthaltend mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a), mindestens eine Nukleinsäure gemäß (b), mindestens einen Vektor gemäß Punkt (c) oder mindestens einen Antikörper gemäß (e)
• (f) mindestens einer Verbindung gemäß einem der Ansprüche 25 oder 26 und/oder
• (g) mindestens eines Wirkstoffs, der an mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a) bindet

zur Behandlung von Schmerz und als weiterer Gegenstand der Erfindung die Verwendung der vorgenannten Moleküle (a) bis (g) zur Herstellung eines Arzneimittels zur Behandlung von Schmerz. Bevorzugt handelt es sich um chronischen, insbesondere neuropathischen oder entzündungsbedingten Schmerz.Another object of the invention is the use

• (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 coded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins,
• (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid,
• (c) at least one vector containing a nucleic acid according to (b),
• (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a),
• (e) at least one cell containing at least one protein or a functional fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (e)
• (f) at least one compound according to one of claims 25 or 26 and / or
• (g) at least one active substance which binds to at least one protein or a functional fragment or a functional variant thereof according to (a) binds

for the treatment of pain and as a further subject of the invention, the use of the aforementioned molecules (a) to (g) for the manufacture of a medicament for the treatment of pain. It is preferably chronic, especially neuropathic or inflammatory pain.

at the use of molecules according to the invention as Medicines for the treatment of pain will be the one to be treated Patients preferably administered recombinantly produced protein. Alternatively, cells are taken from the patient to be transfected, these in vitro with (expression) vectors according to the invention transfected, cultured and then transferred to the patient as a retransplant. The Transfection is preferably by nucleic acids, nucleic acid constructs or (expression) vectors that carry out the expression Pair a regulatable promoter. The transfected self-transplant may be injected locally, for example, depending on the specific disease and the specific target cells.

• (a) mindestens eine Nukleinsäure codierend für mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BUb4, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL oder ein funktionelles Fragment oder eine funktionelle Variante hiervon und/oder mindestens eine Nukleinsäure umfassend oder bestehend aus eine(r) Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 und/oder mindestens eine Nukleinsäure, insbesondere eine Antisense Nukleinsäure oder eine PNA, welche eine Sequenz aufweist, die in der Lage ist, spezifisch an eine der vorgenannten Nukleinsäuren zu binden und/oder ein funktionelles Fragment oder eine funktionelle Variante der mindestens einen Nukleinsäure,
• (b) mindestens eines Vektors enthaltend eine Nukleinsäure gemäß (a), und/oder
• (c) mindestens einer Zelle enthaltend mindestens eine Nukleinsäure gemäß (a) oder mindestens einen Vektor gemäß Punkt (b)

für die Gentherapie.Another object of the invention relates to the use

• (a) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BUb4, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid,
• (B) at least one vector containing a nucleic acid according to (a), and / or
• (c) at least one cell containing at least one nucleic acid according to (a) or at least one vector according to item (b)

for gene therapy.

According to the invention it can these are in vivo or in vitro gene therapy. Under "gene therapy" is a form of To understand the therapy in which by the introduction of nucleic acids in This means, for example, that an effector gene is expressed a defective gene will be replaced or an additional gene introduced. According to the invention it can these are in vivo or in vitro gene therapy. In the in vitro gene therapy, cells are removed from the organism, transfected with vectors in vivo and subsequently returned to the same or to be introduced into another organism. At the in vivo Gene therapy will be vectors, for example, to combat Tumors, systemic (e.g., via the bloodstream) or directly into the target tissue (e.g., a tumor) administered. Preference is for use in gene therapy, the use of a nucleic acid, in which is an antisense nucleic acid or a PNA, or part of a ribozyme, other DNA enzyme or catalytic Represents RNA or DNA.

• (a) mindestens eines Proteins ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL und/oder mindestens eines Proteins umfassend eine Aminosäuresequenz gemäß einer der 11, 18, 20, 27, 29, 31, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 und/oder mindestens eines Proteins, für das eine Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 codiert, und/oder mindestens eines Proteins, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 oder deren Antisense Nukleinsäure hybridisiert oder eines funktionellen Fragments oder einer funktionellen Variante eines der vorgenannten Proteine,
• (b) mindestens eine Nukleinsäure codierend für mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL oder ein funktionelles Fragment oder eine funktionelle Variante hiervon und/oder mindestens eine Nukleinsäure umfassend oder bestehend aus eine(r) Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 und/oder mindestens eine Nukleinsäure, insbesondere eine Antisense Nukleinsäure oder eine PNA, welche eine Sequenz aufweist, die in der Lage ist, spezifisch an eine der vorgenannten Nukleinsäuren zu binden und/oder ein funktionelles Fragment oder eine funktionelle Variante der mindestens einen Nukleinsäure,
• (c) mindestens eines Vektors enthaltend eine Nukleinsäure gemäß (b),
• (d) mindestens eines Antikörpers gegen eines der Proteine oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a),
• (e) mindestens einer Zelle enthaltend mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a), mindestens eine Nukleinsäure gemäß (b), mindestens einen Vektor gemäß Punkt (c) oder mindestens einen Antikörper gemäß (d)
• (f) mindestens einer Verbindung gemäß einem der Ansprüche 25 oder 26 und/oder
• (g) mindestens eines Wirkstoffs, der an mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a) bindet,

für die Diagnostik und/oder für Wirksamkeitsuntersuchungen. Erfindungsgemäß ist unter „Diagnostik" die Analyse von Symptomen, die einem Krankheitsbild zugeordnetet sind, zu verstehen und unter „Wirksamkeitsuntersuchungen" die Untersuchung der Wirksamkeit, insbesondere der medizinischen Wirksamkeit, von Testsubstanzen zu verstehen.Another object of the invention relates to the use

• (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 coded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins,
• (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid,
• (c) at least one vector containing a nucleic acid according to (b),
• (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a),
• (e) at least one cell containing at least one protein or a functional fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (d)
• (f) at least one compound according to one of claims 25 or 26 and / or
• (g) at least one active substance which binds to at least one protein or a functional fragment or a functional variant thereof according to (a),

for diagnostics and / or for efficacy studies. According to the invention, the term "diagnosis" is to be understood as the analysis of symptoms which are associated with a clinical picture, and "effectiveness tests" are to be understood as the investigation of the effectiveness, in particular the medical effectiveness, of test substances.

• (a) mindestens eines Proteins ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL und/oder mindestens eines Proteins umfassend eine Aminosäuresequenz gemäß einer der 11, 18, 20, 27, 29, 31, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 und/oder mindestens eines Proteins, für das eine Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 codiert, und/oder mindestens eines Proteins, das durch eine Nukleinsäure codiert wird, die unter stringenten Bedingungen mit einer Nukleinsäure umfassend eine Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 oder deren Antisense Nukleinsäure hybridisiert oder eines funktionellen Fragments oder einer funktionellen Variante eines der vorgenannten Proteine,
• (b) mindestens eine Nukleinsäure codierend für mindestens ein Protein ausgewählt aus der Gruppe bestehend aus KIAA0378, BAB31214/Q9BU64, OPA1/KIAA0567, CPG2/KIAA1756, LETM1, PID12832121/FLJ20420 und PGRL oder ein funktionelles Fragment oder eine funktionelle Variante hiervon und/oder mindestens eine Nukleinsäure umfassend oder bestehend aus eine(r) Nukleinsäuresequenz gemäß einer der 12, 19, 21, 28, 30, 32, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 und/oder mindestens eine Nukleinsäure, insbesondere eine Antisense Nukleinsäure oder eine PNA, welche eine Sequenz aufweist, die in der Lage ist, spezifisch an eine der vorgenannten Nukleinsäuren zu binden und/oder ein funktionelles Fragment oder eine funktionelle Variante der mindestens einen Nukleinsäure,
• (c) mindestens eines Vektors enthaltend eine Nukleinsäure gemäß (b),
• (d) mindestens eines Antikörpers gegen eines der Proteine oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a), und/oder
• (e) mindestens einer Zelle enthaltend mindestens ein Protein oder ein funktionelles Fragment oder eine funktionelle Variante hiervon gemäß (a), mindestens eine Nukleinsäure gemäß (b), mindestens einen Vektor gemäß Punkt (c) oder mindestens einen Antikörper gemäß (d)

in einem Verfahren zur Auffindung schmerzregulierender Substanzen.Another object of the invention relates to the use

• (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 coded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins,
• (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid,
• (c) at least one vector containing a nucleic acid according to (b),
• (D) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), and / or
• (e) at least one cell containing at least one protein or a functional fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (d)

in a method for finding pain-regulating substances.

The The present invention will be described below with reference to figures and examples illustrated but not limited to these.

characters

1 Figure 2 shows the Coomassie stained BAC / SDS-PAGE gel of separation of the Triton X-100 resistant protein pellet from synaptic membranes of the dorsal spinal cord. The arrows indicate the various protein spots ("spot"). The separation of the proteins described in example 3 led to the identification the proteins KIAA0378 (see Spot21) and PID12832121 / FLJ20420 (see spot 76).

2 shows the peptide mass fingerprint of KIAA0378. Shown is the evaluation of the peptide mass fingerprints using the program ProFound (http://129.85.19.192/profound_bin/WebProFound.exe). The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

Measured Mass (M):

gemessene Peptidmasse

Computed Mass:

berechnete Masse

Error(ppm):

Abweichung der gemessenen von der berechneten Masse in parts per million.

Avg/Mono:

Bemittelte oder monoisotope Peptidmasse; für die Datenbanksuche wurden ausschließlich monoisotope Peptidmassen bestimmt (indiziert in der Auswertung durch M).

Residues Start-To:

Position der Aminosäurereste in der Proteinsequenz, die zu dem Peptid gehören, das einer gemessenen Peptidmasse innerhalb der gesetzten Fehlertoleranz entspricht.

Missed Cut:

von der Protease überlesene Spaltstellen

Peptide Sequence:

Sequenz des Peptides, dessen Masse mit einer gemessenen Masse übereinstimmt.

(1)+O@M:

Die gemessene Masse stimmt mit dem vorgeschlagenen Peptid dann überein, wenn der Methioninrest oxidiert vorliegt.

Sequenzabdeckung:

prozentualer Anteil der Gesamtsequenz des Kandidatenproteins, der mit den passenden gemessenen Peptidmassen abgedeckt wird.

The following is the legend of the peptide mass allocation tables, this applies to 2 as well as for the following ones 3 . 5 . 6 . 7 . 9 and 10 ,

Measured Mass (M):

measured peptide mass

Computed Mass:

calculated mass

Error (ppm):

Deviation of the measured from the calculated mass in parts per million.

Avg / Mono:

Averaged or monoisotopic peptide mass; for the database search only monoisotopic peptide masses were determined (indicated in the evaluation by M).

Residuals Start-To:

Position of amino acid residues in the protein sequence belonging to the peptide corresponding to a measured peptide mass within the set fault tolerance.

Missed Cut:

cleavage sites read by the protease

Peptide Sequence:

Sequence of the peptide whose mass matches a measured mass.

(1) + O @ M:

The mass measured is in agreement with the proposed peptide when the methionine residue is oxidized.

Sequence coverage:

percentage of the total sequence of the candidate protein covered with the appropriate measured peptide masses.

3 shows the peptide mass fingerprint of PID12832121 / FLJ20420 putative protein. Shown is the evaluation of the peptide mass fingerprints using the program ProFound (http://129.85.19.192/profound_bin/WebProFound.exe). The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

4 Figure 2 shows the Coomassie stained BAC / SDS-PAGE gel of separation of the chaotrope-resistant protein pellet from synaptic membranes of the dorsal spinal cord. The arrows indicate the different protein spots. The separation of the proteins described in Example 3 led to the identification of the proteins LETM1 (see Spot19), OPA-1 / KIAA0567 (see Spot22) and PGRL (see Spot23).

5 shows the peptide mass fingerprint of the transmembrane protein LETM1. The evaluation of the peptide mass fingerprints using the program ProFound (http://129.85.19.192/profound_bin/WebProFound.exe) is shown. The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

6 shows the peptide mass fingerprint of OPA-1 / KIAA0567. Shown is the evaluation of the peptide mass fingerprints using the program ProFound (http://129.85.19.192/profound_bin/WebProFound.exe). The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

7 shows the peptide mass fingerprint of the immunoglobulin superfamily receptor PGRL. Shown is the evaluation of the peptide mass fingerprints using the program ProFound (http://129.85.19.192/profound_bin/WebProFound.exe). The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

8th Figure 4 shows the Coomassie-stained BAC / SDS-PAGE gel of separation of the chaotrope-resistant protein pellet from rat / formalin dorsal spinal cord synaptic membranes. The arrows indicate the different protein spots. The separation of the proteins described in Example 3 led to the identification of the proteins CPG2 (see Spot29) and BAB31214 / Q9BU64 (see Spot94).

9 shows the peptide mass fingerprint of CPG2 / KIAA1756 (script: CPG2). Shown is the evaluation of the peptide mass fingerprints using the program ProFound (httpt: //129.85.19.192/profound_bin/WebProFound.exe). The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

10 shows the peptide mass fingerprint of the putative protein BAB31214. Shown is the evaluation of the peptide mass fingerprints using the program ProFound (http://129.85.19.192/profound_bin/WebProFound.exe). The NCBI non-redundant protein database (last updated November 2002) was searched. The gene product was identified as positive according to the program's significance criteria. The positively associated peptide masses are shown.

The 11 to 17 concern the protein KIAA0378:

11 shows the amino acid sequence of the human protein KIAA0378

12 shows the nucleic acid sequence of the human KIAA0378 mRNA

13 shows the result of the Northern blot analysis for the expression of KIAA0378 mRNA in the adult rat. As can be seen, expression of the mRNA could only be observed in the brain. A smaller size transcript was detected in the kidney, which could be an alternative transcript or a closely related gene. In all other tissues examined (heart, lung, liver, kidney, spleen, stomach, small intestine, thymus, skeletal muscle, testis, uterus, placenta) no expression was observed. These findings suggest a selective expression of KIAA0378 in the nervous system and suggest that the use of KIAA0378 in the development of side-effect analgesics is very interesting.

14 shows the cellular localization of the KIAA0378 mRNA transcript in adult rat dorsal root ganglia by in situ hybridization. There is a strong signal of KIAA0378 mRNA in the DRGs. A detailed assignment to individual cell populations was not possible.

15 shows the cellular localization of the KIAA0378 mRNA transcript in the adult rat spinal cord by in situ hybridization. It can be seen that the KIAA0378 mRNA was not detected in the spinal cord.

16 and 17 show the cellular localization of the KIAA0378 mRNA transcript in the brain (hippocampus) ( 16 ) and in the total brain ( 17 ) of the adult rat by in situ hybridization. As can be seen, the KIAA0378 mRNA could be detected in the following brain regions: olfactory nucleus, cortex, hippocampus (CA3, denate gyrus), substantia nigra, brainstem nuclei (pontine nuclei), plexus chorioideus.

The 18 to 26 concern the protein BAB31214 / Q9BU64:

18 shows the amino acid sequence of the human protein Q9BU64

19 shows the nucleotide sequence of the human Q9BU64 gene (Accession No. BC 002870)

20 shows the amino acid sequence of the mouse orthologue BAB31214 to the human protein Q9BU64

21 shows the nucleotide sequence of the mouse orthologue BAB31214 gene to the human Q9BU64 gene

22 Figure 2 shows Northern blot analysis for expression of adult rat BAB31214 / Q9BU64 mRNA (probe BAB 21974) representing the rat orthologues to human Q9BU64 and BAB31214, respectively. It can be seen that only in the brain and in the spleen expression of the mRNA are observed, in the testes a transcript smaller size was detected, this could be an alternative transcript or a closely related gene. In all other tissues examined (heart, lung, liver, kidney, stomach, small intestine, thymus, skeletal muscle, uterus, placenta) no expression was observed. These findings suggest a selective expression of Q9BU64 / BAB31214 in the nervous system and suggest that the use of BAB31214 for the development of low-side-effects analgesics is very interesting.

23 shows the cellular localization of the BAB31214 / Q9BU64 mRNA transcript in adult rat dorsal root ganglia by in situ hybridization. It can be seen that the BAB31214 / Q9BU64 mRNA had a very strong signal in the DRGs. A detailed assignment to individual cell populations was not possible in the present analysis.

24 shows the cellular localization of the BAB31214 / Q9BU64 mRNA transcript in the adult rat spinal cord by in situ hybridization. As a result, the BAB31214 / Q9BU64 mRNA in the spinal cord showed a very strong signal in the gray matter, both in the deep dorsal horn and in the anterior horn. Motor neurons show very strong expression.

25 and 26 shows the cellular localization of the BAB31214 / Q9BU64 mRNA transcript in the brain ( 25 ) and total brain ( 26 ) of the adult rat by in situ hybridization. The BAB31214 / Q9BU64 mRNA could be detected in the following brain regions: olfactory nucleus, cortex, hippocampus (CA1-3, DG dentate gyrus), caudate nucleus and putamen (striatum), ventral palidum, thalamus (Nucl.thal.mediodorsal , central medial and centrolateralis, dorsal cortex of the colliculi of the tectum), hypothalamus (arcuate hypothal Nucl., dorsomedial hypothalic Nucl.) Substantia nigra, zona incerta, brainstem nuclei (pontine nuclei, 7 facial nuclei, reticular nuclei), choroid plexus, cerebellum.

The 27 to 37 concern the protein OPA-1 / KIAA0567:

27 shows the amino acid sequence of the human OPA / 1KIAA0567 protein

28 shows the nucleotide sequence of the human OPA-1 / KIAA0567 mRNA

29 shows the amino acid sequence of mouse OPA-1 / KIAA0567 protein

30 shows the nucleotide sequence of mouse OPA-1 / KIAA0567 mRNA

31 shows the amino acid sequence of the rat OPA-1 / KIAA0567 protein

32 shows the nucleotide sequence of the rat OPA-1 / KIAA0567 mRNA

33 Figure 2 shows Northern blot analysis for expression of OPA-1 / KIAA0567 mRNA in adult rat (probe BB617042). It can be seen that the strongest expression in the brain was present, followed by the placenta and the uterus. In all other tissues examined (heart, lung, liver, kidney, spleen, stomach, small intestine, thymus, testes, skeletal muscle) no expression was observed. These findings suggest a relatively selective expression of OPA-1 / KIAA0567 in the nervous system.

34 shows the cellular localization of the OPA-1 / KIAA0567 mRNA transcript in adult rat dorsal root ganglia by in situ hybridization (probe BB617042). The OPA-1KIAA0567 mRNA had a relatively weak signal in the DRGs. A detailed assignment to individual cell populations was not possible in the present analysis.

35 Figure 2 shows the cellular localization of the OPA-1 / KIAA0567 mRNA transcript in the adult rat spinal cord by in situ hybridization (probe BB617042). The OPA-1 / KIAA0567 mRNA showed a relatively weak signal in the spinal cord in the gray matter with the strongest signal in the superficial posterior horn (DH) in the laminae I / II. In addition, signals were shown in the motor neurons.

36 and 37 shows the cellular localization of the OPA-1 / KIAA0567 mRNA transcript brain ( 37 ) and in the total brain ( 38 ) of the adult rat by in situ hybridization. The OPA-1 / KIAA0567 mRNA could be detected in the following brain regions: Olfactory nu, cortex, dorsal cortex colliculus, hippocampus (CA1-3, DG dentate gyrus), caudate nucleus + putamen (striatum), hypothalamus, amygdala, cerebellum.

The 38 to 43 concern the protein CPG2 / KIAA1756:

38 shows the amino acid sequence of the human KIAA1756 protein

39 shows the nucleotide sequence of the human KIAA1756 mRNA

40 shows the amino acid sequence of mouse CPG2 protein

41 shows the nucleotide sequence of mouse CPG2 mRNA

42 shows the amino acid sequence of the CPG2 rat protein

43 shows the nucleotide sequence of the CPG2 rat mRNA

The 44 to 49 concern the protein LETM1:

44 shows the amino acid sequence of the human LETM1 protein

45 shows the nucleotide sequence of the human LETM1 mRNA

46 shows the amino acid sequence of the mouse LETM1 protein

47 shows the nucleotide sequence of the mouse LETM1 mRNA

48 shows the amino acid sequence of the rat LETM1 protein

49 shows the nucleotide sequence of the rat LETM1 mRNA

The 50 to 55 concern the protein PID12832121 / FLJ20420:

50 shows the amino acid sequence of the human FLJ20420 protein

51 shows the nucleotide sequence of the human FLJ20420 mRNA

52 shows the amino acid sequence of mouse FLJ20420 protein

53 shows the nucleotide sequence of mouse FLJ20420 mRNA

54 shows the amino acid sequence of the rat PID12832121 protein

55 shows the nucleotide sequence of the rat PID12832121 mRNA

The 56 to 61 concern the protein PGRL:

56 shows the amino acid sequence of the human PGRL protein

57 shows the nucleotide sequence of the human PGRL mRNA

58 shows the amino acid sequence of the mouse PGRL protein

59 shows the nucleotide sequence of the mouse PGRL mRNA

60 shows the amino acid sequence of the rat PGRL protein

61 shows the nucleotide sequence of the rat PGRL mRNA

Examples

Example 1: Preparation synaptic membranes (according to Tom Dieck et al., 1998)

Starting material were dorsal halves from rat spinal cord. The tissue was first taken up in preparation buffer I (0.32 M sucrose, 5 mM HEPES / NaOH pH 7.4, protease inhibitors) to 10 ml per gram wet weight of tissue and homogenized in a glass / Teflon homogenizer (12 puffs, 900 rpm). The homogenate was centrifuged for 10 min at 1000 x g. The supernatant was repealed for reuse, the pellet resuspended in buffer I. The homogenization procedure was repeated, after centrifugation the supernatant was combined with the first supernatant (supernatants S1). The S1 fraction was centrifuged for 15 min at 12,000 x g. The pellet was taken up in buffer I for further processing. Homogenization was repeated (6 puffs, 900 rpm) and centrifugation at 12,000 x g for 20 min. The pellet was added to 1.5 ml per gram wet weight of starting tissue in buffer II (0.32 M sucrose, 5 mM Tris / HC1, pH 8.1, protease inhibitors) and loaded onto a discontinuous sucrose gradient with layering (from bottom to top): 1.2 M sucrose, 1 M sucrose, 0.8 M sucrose. Density gradient centrifugation was performed for 2 hours at 85,000 x g. The synaptosomal fraction could be harvested at the phase boundary of 1.2M sucrose / 1M sucrose. The synaptosomes were exposed to osmotic shock (five volumes of 1 mM Tris / HCl, pH 8.1 with stirring at 0 ° C for 30 min). The synaptosomal membranes were pelleted by centrifugation at 33,000 x g for 30 min.

Example 2: Subfractionation of synaptosomal membranes (Dreger et al. strategy, 2001)

After protein determination (Bradford method, 1976: Bradford M .: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principles of protein-dye binding., Anal. Biochem., 72, 248-254) were each 200 .mu.g of the Prepared crude synaptic membranes used for further fractionation. The preparation was either (a) adjusted to 0.5% (w / v) Triton X-100 and shaken for 15 min at 4 ° C, then centrifuged for 15 min in a bench top centrifuge, or (b) centrifuged for 10 min in the bench top centrifuge, and taken up in 4M urea / 0.1M Na ₂ CO ₃ solution according to the starting volume. After resuspension, the preparation was centrifuged off for 30 min in a tabletop ultracentrifuge (Airfuge, Beckman-Coulter GmbH, Krefeld at 2.8 psi). Both after procedure (a) and after procedure (b), the pellet fractions were further processed. The subfractionation enriched different substructures of synaptic membranes and partially made non-overlapping protein populations accessible to analysis.

Example 3: Separation of proteins and preparation for the mass spectrometry

The Proteins were placed in sample buffer for 16-benzyldimethyl-hexadecylammonium chloride (16-BAC) gel electrophoresis recorded and separated on 4-10% acrylamide gradient gels. In a second separation, the proteins were then in an SDS-PAGE separated. The method according to Hartinger et al., 1996, was requested References applied later. The protein gels were with Coomassie G-250 dyed. All distinguishable protein spots were cut out manually. The proteins were gelated by the method of Shevchenko et al. (1996) split. The peptide mixtures were analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). The proteins were database-aided by identified the programs ProFound and Mascot. In some cases, individual Peptides selected, fragment ion spectrum for obtaining partial sequence information take.

Example 4: Northern blot Analyzes for creating a mRNA expression profile in different Tissues of the adult rat

to Generation of an mRNA expression profile of the identified proteins In various tissues of the adult rat were Northern blot Analyzes performed under the following conditions:

Radioactive label of the cDNA fragment

• (Ready-To-Go DNA Labeling Kit, Amersham Pharmacia)
• - 50ng cDNA in 45μl Dilute water
• - 3 min at 100 ° C denature,
• - on Shake off ice
• - too Add to the DNA Labeling Bead
• - careful Pipette up and down until the bead has completely dissolved
• Addition of 5 μl [α- ³³ P] dCTP 10mCi / ml [50 μCI] (Hartmann Analytics, Brunswick)
• - again Carefully pipette up and down
• - 1 h at 37 ° C incubate
• - 2 min on ice
• - short Spin down
• - Probe via ProbeQuant Purify G-50 Micro Column (Amersham Pharmacia):
• - 3 Centrifuge at 2800 rpm
• - probes Apply in the middle
• - 3 Centrifuge at 2800 rpm
• - Add 1μl in 15 ml scintillator fluid: ³³ P measurement on LKB Counter
• - Target activity: 1-2 x 10 ⁶ cpm / ml hybridization solution

Prehybridization of the filters

• Add 5ml Micro-Hyb ^™ buffer (Invitrogen) to the filters
• + 5 μg COT-1 DNA (Invitrogen) (previously denatured at 100 ° C for 3 min)
• + 5 μg Poly dA (Roche)
• at 42 ° C in the hybridization oven for Prehybridize for 2 hours

Hybridization of the filters

• ^{Denature 33} P DNA fragment for 3 min at 100 ° C
• put off on ice
• add to the prehybridization solution (should: 1-2 x 10 ⁶ cpm / ml hybridization solution) hybridize overnight at 42 ° C (approximately 12-18 hrs)

Washing the filters

• 2 × 20 min with 2 × SSC / 1 % SDS at 50 ° C
• 1 × 15 min with 0.5 × SSC / 1 % SDS at 55 ° C

Evaluation of the filters

• Exposure for 2-7 days with ImageScreen (Fuji)
• Measurement of the screens with the StormImager ^TM (Molecular Devices)

Example 5: In situ hybridization to the cellular Localization of mRNA transcripts in the nerve tissue of the rat

to Analysis of the cellular Localization of the mRNA of the identified proteins in pain-relevant tissues The adult rat underwent in situ hybridization experiments following conditions.

cRNA samples

Specific fragments of the cDNAs examined were subcloned into the vector pCRII-TOPO and used for in vitro transcription. Transcription using ³⁵ S-UTP and purification of the transcripts was performed according to Schäfer et al. (1994). After transcription, the samples were subjected to moderate alkaline hydrolysis as described by Angerer et al. (1987).

In situ hybridization

The in situ histochemical analysis was carried out on the basis of an already published protocol (Schäfer et al., 1992, Schäfer & Day, 1995). Here, cryostat sections were fixed with a thickness of 12 .mu.m in 4% formaldehyde solution for one hour and then washed three times for 10 minutes with 10mM phosphate buffered solution (PBS), pH7.4. A final washing step was carried out with 10 mM PBS with 0.4% Triton X-100. The sections were rinsed briefly with distilled water and washed with 0.1M triethanolamine solution pH 8.0 followed by a wash with 0.1M triethanolamine pH 8.0 with 0.25% (vol / vol) acetic anhydride for 10 min at room temperature. After incubation in a 2xSSC buffer, the sections were dehydrated in an ascending alcohol series (50%, 70%) and air-dried. The radioactive samples in the hybridization solution (3X SSC, 50 mM NaPO ₄ , 10 mM dithiothreitol, 1 × Denhardt's solution, 0.25 g / L yeast tRNA, 10% dextran sulfate and 50% formamide) were added to a final concentration of 5 × 10 ⁴ dpm / μl diluted. 30-50μl of this solution was added to each tissue section, covered with a cover slip and incubated for 14 hours at 60 ° C. The sections were then washed in 2 x SSC and 1 x SSC for 20 min each, followed by incubation in an RNase buffer (10 mM Tris, pH 8.0, 0.5 M NaCl, 1 mM EDTA) with 1 U / ml RNase T1 and 20 μg / ml RNaseA (Roche, Mannheim) for 30 min at 37 ° C. Consecutive washes were followed in 1X SSC, 0.5X SSC, 0.2X SSC, and distilled water. The sections were dehydrated by incubation in 50% and 70% isopropanol. The composition and preparation of the standard buffers used is described in Sambrook et al. (2001, Volume 3, Appendix 1: A.1.2-A 1.20).

image analysis

The Autoradiography of the 35S-labeled tissue sections was performed by Immerse yourself in a "NTB-2 Nuclear Emulsion "(Eastman Kodak, Rochester, NY). Exposure times were between 10 and 20 days. After photographic development, the autoradiograms were with an Olympus AX_70 microscope and the MCID M4 image analysis software (Imaging Research Inc., Ontario, Canada). A cresyl violet stain was as a counterstain carried out.

Example 6: mRNA Regulation in dorsal root ganglia of rats from chronic pain models

To analyze the regulation of KIAA0378 mRNA in animal pain models (rats) a PIQOR ^™ cDNA array analysis was performed. For this purpose, spinal ganglia are taken from pain-treated animals and control animals and analyzed for gene expression. The procedure is described below:

6.1. PIQOR ^™ cDNA arrays

• – keine repetitiven Elemente (z.B. Alu, B1, MIRs, Mikrosatelliten) enthalten,
• – Sequenzhomologie zu allen anderen bekannten cDNAs (öffentliche Datenbanken) < 85%,
• – Die Fragmentlänge: 200 bis 400 Basenpaare (bp),
• – ausgewähltes Fragment deckt jeweils alle alternativen Spleiß- und Polyadenylierungsvarianten ab.

A collection of cDNA fragments (human, mouse, rat species) specifically designed to produce PIQOR ^™ cDNA arrays was prepared. In order to achieve the highest possible selectivity and to avoid false positive or false negative results due to cross-hybridization, cDNA fragments were prepared according to the following criteria:

• - contain no repetitive elements (eg Alu, B1, MIRs, microsatellites),
• Sequence homology to all other known cDNAs (public databases) <85%,
• The fragment length: 200 to 400 base pairs (bp),
• Each fragment selected covers all alternative splicing and polyadenylation variants.

6.2. Production of topic-specific PIQOR ^TM arrays

PIQOR ^™ arrays were made with a total of 1241 cDNAs. 636 cDNAs are PIQOR ^™ fragments (species rat, including 64 mouse fragments), 583 cDNAs were isolated by subtractive hybridization (GT clones) and 16 cDNAs were from other sources. As positive controls, six housekeeping genes and four DNA fragments from E. coli (CR: control RNA) were applied to the arrays. The latter were added to the rat RNA in the form of in vitro transcribed RNA to control the labeling and hybridization process. Salmon sperm DNA and buffer were applied as negative controls. From each cloned cDNA fragment, amplification of the insert was carried out with vector primers, the amplificates were separated in an agarose gel and the length of the inserts was controlled. The amplified cDNAs were then purified and adjusted to a uniform concentration of about 100 ng / μl. Equal amounts of the PCR amplicons of the cloned cDNA fragments were spotted onto the surface of derivatized slides using a dispenser. For quality control, the DNA of the produced batch was stained with a fluorescent dye. Each cDNA was applied four times, giving a total of 4964 spots per array.

6.3. Preparation of the Dorsal root ganglia

There was a treatment of five animals per condition as well as the removal of the dorsal root ganglia. For hybridization, amplified RNA (aRNA) from performed preparations of the PIQOR ^™ fragments of shock frozen tissue was used.

6.4. Hybridization of the PIQOR ^™ cDNA arrays

For the determination of the expression profiles, in each case 2 μg of aRNA from the test group were labeled with Cy5 and the same amount of aRNA from the control group was hybridized with Cy3 and hybridized together on a PIQOR ^™ cDNA array. Labeling was achieved by reverse transcription with incorporation of fluorescent nucleotides (Cy5-dCTP and Cy3-dCTP, respectively). Tables 1 and 2 give an overview of the hybridizations performed.

Table. Figure 1: Summary of the first nine hybridizations on the PIQOR ^™ cDNA arrays

Table. Figure 2: Summary of the other five hybridizations on the PIQOR ^™ cDNA arrays

Results of the cDNA array analyzes

For the cDNA fragment The KIAA0378 protein became one in the Chung model 7 days after surgery Expression was observed, however, with a quotient of 1.01 no regulation detectable. In all other models studied (see Table 1 and 2) no expression was detected.

The experiment was carried out using the PIQOR ^™ array hybridization protocol (see below under I.-VII.). Excerpts from the PIQOR ^TM Array Hybridization Protocol are listed below:

I. mRNA amplification protocol

I.1 first-strand synthesis

The following substances were combined in a 1.5 ml RNase-free microcentrifuge tube:
4 μl 5 × first strand buffer (Gibco)
2 μl 0.1 M DTT (Gibco)
1 μl of 100 μM T7 (T) 24 primer
1 μl of 10 mM dNTPs

10 μl of total RNA were added (with recommended 2 μg of total RNA as Starting material for to use the amplification). Then (optionally with high speed) mixed. The mixture was stirred at 65 ° C for 5 min. incubated. Then the tube became ("tube") stored at 42 ° C for 2 min. After all 2 μl SSII RT (400 U) (Gibco) added. It had to be ensured that the enzyme was in a well mixed Reaction added has been. The incubation was carried out at 42 ° C for 1 hour and then put the incubated material on ice.

I.2 Second-strand synthesis

All reagents and strand "tubes" were iced. It was added to the strand "tubes":
91.3 μl DEPC H2O
30 μl 5 × second strand buffer (Gibco)
4 μl of DNA polymerase I (40 U) (Gibco)
3 μl of 10 mM dNTPs
1 μl of E. coli DNA ligase (10 U) (Gibco)
0.7 μl RNase H (2 U) (Gibco)

It was heavily mixed (at high speed). At 16 ° C was incubated for 2 hours and optional stored at -20 ° C.

I.3 Purification ("clean-up") of the ds cDNA (see also QIAquick · Spin Handbook)

750 μl of buffer PB was added to the strand "tube". A QIAquick spin column was placed in a 2 ml collection tube. The sample was applied to the QIAquick column. It was centrifuged for 1 min. The flow was discarded and the QIAquick column set back into the same "tube". 0.75 ml of buffer PE was added to the QIAquick column. It was centrifuged for 1 min. The flow was discarded and the QIAquick column set back into the same "tube". The "tube" was centrifuged for a further minute at maximum speed. The QIAquick spin column was placed in a clean 1.5 ml tube To elute the ds cDNA, 30 μl of RNase-free H ₂ O was added to the center of the QIAquick membrane Incubated for 1 min at room temperature, then centrifuged at maximum speed for 1 min and finally concentrated to 8 μl.

I.4 aRNA synthesis: T7 in vitro transcription (Ambion)

All reagents were thawed and brought to room temperature. It should be noted that spermidine in the transcription buffer can lead to a precipitate of the template DNA when the reaction takes place on ice. An NTP mix (per "tube") was produced:
2 μl ATP solution (75 mM)
2 μl GTP solution (75 mM)
2 μl CTP solution (75 mM)
2 μl UTP solution (75 mM)
2 μl 10x reaction buffer

Then "tube" (purified) was added to the ds-cDNA:
10 μl NTP mix
2 μl enzyme mix (Ambion)

It was mixed moderately (at low speed) and at 37 ° C for 6 hours incubated. It should be noted that the IVT "tubes" in a water bath constant temperature or a thermocycler with a heating "lid" (0.2-ml "tube") should.

I.5 In Vitro Transcription "Clean-up" (see RNeasy "Mini Handbook)

It was mixed in an IVT reaction tube:
80 μl RNase-free H2O
350 μl RLT buffer

Then 250 μl 100% EtOH added. This sample was transferred to the RNeasy spin column. It was for 15 Sec rotated at maximum speed. The transfer spin column was to a new "collection tube "transferred.

500 μl RPE buffer was added. It was shot at maximum speed for 15 sec. The spin column was transferred to a new collection tube. 500 μl RPE buffer was added. It was rotated for 2 min. At maximum speed. The spin column was transferred to a new collection tube. 50 μl of RNase-free H ₂ O was added to the membrane of the spin column and incubated for 4 min. It was rotated for 1 min. At maximum speed. Steps 13-15 were repeated using elution as elution. Thereafter, the OD (1:50 dilution) was measured. Optionally, storage at -20 ° C take place.

II. Labeling reaction

The following substances were combined in a 1.5 ml RNase-free microcentrifuge tube:

It were 15 μl the aRNA (2 μg) and 2 μl each added to control RNA 1 and 2. Then was vortexed, with high speed. At 65 ° C was for Incubated for 5 min and then the incubation solution was on 42 ° C brought. 1 μl (200 u) of the SSII enzyme, while ensuring that the enzyme mixed well, the reaction was added. At 42 ° C was for 30 min. incubated. Then 2 μl of the SSII enzyme (again the enzyme is well mixed with the reaction added should be added), then at 42 ° C for 30 Min. Incubated, then 0.5 .mu.l RNase H added. At 37 ° C was for Incubated for 20 min to hydrolyze RNA.

III. Samples "clean-up" (see QIAquick 'Spin Handbook)

Cy3 and Cy5 labeled samples were combined and 400 μl of buffer PB added. A QIAquick spin column was added to a 2 ml "collection tube". The sample was applied to the QIAquick column and centrifuged for 30-60 sec. The flow was discarded. The QIAquick column was returned to the same "tube". To wash, 0.75 ml of buffer PE was added to the QIAquick column and centrifuged for 30-60 sec. The flow was discarded and the QIAquick column returned to the same "tube". The column was centrifuged for a further minute at maximum speed. The QIAquick column was placed in a sterile ("clean") 1.5 ml "microfuge tube". To elute the DNA, 30 μl of H ₂ O pH 8 was added to the center of the QIAquick membrane. The column was allowed to stand for 1 min and then centrifuged for 1 min. It was concentrated in a "SpeedVac" at 45 ° C to a volume of ~ 10μl, taking care that the sample does not dry out. 10 μl of 2 × hybridization solution prewarmed to 42 ° C was added. The sample was stored in the dark at room temperature until pre-hybridization was complete.

IV. PIQOR ^™ Chip Pretreatment

The PIQOR ^™ chip was heated at 98 ° C in distilled water for 2 min. The PIQOR ^™ chip was transferred to 96% ethanol and dried by centrifugation at 500 x g for 3 min and placed in a dust-free cassette.

V. Pre-hybridization

The PIQOR ^™ pre-hybridization solution was heated at 98 ° C for 2 min, spun quickly and cooled to 42 ° C. The PIQR ^™ chip was placed on the "Pattern Slide". 20 μl of the PIQOR ^™ Pra hybridization solution was applied to the given rectangle of the PIQOR ^™ chip. The coverslip was placed on the PIQOR ^™ chip and the chip was inserted into the moistened hybridization cassette (eg PIQOR ^™ - HybChamb), the hybridization cassette was sealed and incubated at 62 ° C for at least 30 min.

VI. hybridization

The hybridization cassette was cooled to 25 ° C. The PIQOR ^™ chip was placed on the "Pattern Slide". The coverslip was carefully removed from the PIQOR ^™ chip. 20 μl of the labeled sample was applied to the given rectangle of the PIQOR ^™ chip. The coversheet was placed on the PIQOR ^™ chip (the cover slip of the pre-hybridization should not be reused) and placed in the humidified hybridization cassette and incubated at 62 ° C for at least 6 hours.

VII. Washing

The PIQOR ^™ chip was removed from the hybridization cassette (cooling should be avoided!) And immediately placed in a wash bowl filled with 1X Wash Buffer 1 for 5 min (room temperature 20-30 ° C). The washing step was repeated with fresh 1X Wash Buffer 1. The washing step was repeated with 1 × wash buffer 2. It was dried by centrifugation at 500 x g for 3 min. Then it was transferred to a dust-free cassette.

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SEQUENCE LISTING

concordance table

Claims (41)

A method for detecting pain-regulating substances, comprising the following steps: (a) incubation of a substance to be tested under suitable conditions with a cell and / or a preparation from such a cell which contains at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and / or at least one protein. comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a Nucleic acid sequence according to any one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 encoded, and / or a protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense has hybridized nucleic acid or synthesized a functional fragment or a functional variant of one of the aforementioned proteins, (b) measuring the binding of the test substance to the at least one protein synthesized by the cell or functional fragment or functional derivative thereof or measuring at least one of them Binding of the test substance to the protein or functional fragment or functional derivative thereof altered functional parameter. Method according to claim 1, characterized in that the measurement of the bond over the displacement a known labeled ligand of the protein or a functional Fragment or a functional variant thereof and / or on the attached activity a marked test substance takes place. Method according to one of claims 1 or 2, characterized in that the measurement of at least one modified by the test substance functional parameter via measurement of the regulation, inhibition and / or activation of receptors, ion channels and / or enzymes takes place, in particular via measurement of the change in gene expression , the ion milieu, the pH, the membrane potential, the enzyme activity or the concentration of the 2 ^nd messengers. Method according to one the claims 1 to 3, characterized in that the cell before step (a) genetically engineered. Method according to claim 4, characterized in that the genetic engineering manipulation Measurement of at least one of the modified by the test substance functional parameter allowed. Method according to claim 5, characterized in that by the genetic engineering manipulation expressed in the cell not endogenously expressed form of a G protein or a reporter gene is introduced. Method according to one of claims 4 to 6, characterized in that the cell is genetically engineered so that the cell is at least one nucleic acid according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or a functional fragment or a functional variant thereof. Method according to claim 7, characterized in that the nucleic acid in a recombinant DNA construct, preferably a vector, particularly preferably an expression vector, is included. Method according to one the claims 4 to 8, characterized in that the genetically engineered Cell prior to step (a) according to claim 1 under conditions which allow expression to be cultivated, preferably under selection pressure. Method according to one the claims 1 to 7, characterized in that the cell is an amphibian cell, Bacterial cell, yeast cell, insect cell or immortalized or native mammalian cell is. A protein comprising or consisting of an amino acid sequence according to any one of 11 . 20 or 54 or a functional fragment or a functional variant thereof. Protein encoded by a nucleic acid according to any one of 12 . 21 or 55 , A protein encoded by a nucleic acid capable of stringent conditions with a nucleic acid according to any one of 12 . 21 or 55 or whose antisense nucleic acid hybridizes. Nucleic acid, coding for a protein selected from the group consisting of human KIAA0378, murine PID12832121 and rats BAB31214 / Q9BU64 or a functional fragment or a functional variant thereof. Nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 21 or 55 or a functional fragment or a functional variant thereof. Nucleic acid, characterized in that it is DNA, in particular cDNA, or RNA, in particular mRNA act. Nucleic acid, characterized in that it is double or single-stranded nucleic acid. Vector containing a nucleic acid according to one of claims 14 to 17th A cell containing a protein according to any one of claims 11 to 13, a nucleic acid according to one of claims 14 to 17 and / or a vector according to claim 18. Cell according to claim 18, characterized in that the cell is an amphibian cell, Bacterial cell, yeast cell, insect cell or immortalized or native mammalian cell is. antibody against a peptide or protein according to any one of claims 11 to 13th Antisense nucleic acid comprising or consisting from a nucleic acid sequence, which is capable of specific to a nucleic acid according to any one of claims 14 to 17 or a functional fragment or a functional variant to bind from this. PNA comprising or consisting of a nucleic acid sequence, which is capable of specific to a nucleic acid according to any one of claims 14 to 17 or a functional fragment or a functional variant to bind from this. Ribozyme comprising a nucleic acid sequence capable of is specific to a nucleic acid according to a the claims 14 to 17 or a functional fragment or a functional one To bind variant thereof. siRNA, comprising or consisting of a nucleic acid sequence, which is capable of specific to a nucleic acid according to any one of claims 14 to 17 or a functional fragment or a functional variant to bind from this. Aptamer, comprising or consisting of a nucleic acid sequence, which is capable of specific to a protein according to one the claims 11 to 13 or a functional fragment or a functional one To bind variant thereof. Transgenic non-human mammal containing at least a protein according to one of claims 11 to 13, at least one nucleic acid according to one the claims 14 to 17, at least one vector according to claim 18 and / or at least a cell according to any one of claims 19 or 20. A transgenic non-human mammal according to claim 27, characterized characterized in that its germinal and somatic cells pass through the nucleic acid Chromosomal introduction into the genome of the animal or into the genome one of the ancestors of the said animal included. A transgenic non-human mammal according to claim 27 or 28, characterized in that its germinal and somatic cells the nucleic acid by chromosomal manipulation of the genome of the animal or genome one of the ancestors of the named animal in no longer expressible Form included. A transgenic non-human mammal according to any one of claims 27 to 29, characterized in that it is a rodent, in particular a Rat or a mouse, is. Compound identifiable by a method according to one the claims 1 to 10. A compound according to claim 31, characterized in that the compound by a method according to a the claims 1 to 10 is identifiable as a pain-regulating substance. Medicaments containing at least (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 encoded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins, (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1 , PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is able to specifically bind to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, c) at least one vector containing a nucleic acid according to (b), (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), (e) at least one cell containing at least one protein or a functional one A fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to item (c) or at least one antibody according to (d) (f) at least one compound according to one of claims 25 or 26 and / or (g) at least one active ingredient which binds to at least one protein or a functional fragment or a functional one Variant thereof according to (a) binds, and optionally suitable auxiliaries and / or additives. Diagnostic agent containing at least (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 encoded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins, (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1 , PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is able to specifically bind to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, c) at least one vector containing a nucleic acid according to (b), (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), (e) at least one cell containing at least one protein or a functional one A fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to item (c) or at least one antibody according to (d) (f) at least one compound according to one of claims 25 or 26 and / or (g) at least one active ingredient bound to at least one protein or a functional fragment or a radio tional variant thereof according to (a) binds, and optionally suitable additives. Use of (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 coded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins, (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1 , PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 ; 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is able to specifically bind to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, c) at least one vector comprising a nucleic acid according to (b), (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), (e) at least one cell containing at least one protein or a functional one A fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (e) (f) at least one compound according to one of claims 25 or 26 and / or (g) at least one active ingredient which binds to at least one protein or a functional fragment or a function The variant of this according to (a) binds to the treatment of pain. Use of (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein encoded by a nucleic acid comprising a nucleic acid sequence according to one of the Figures V, and / or at least one protein encoded by a nucleic acid which under stringent conditions comprises a nucleic acid comprising a nucleic acid sequence according to any one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins, (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1 , PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is able to specifically bind to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, c) at least one vector comprising a nucleic acid according to (b), (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), (e) at least one cell containing at least one protein or a functional one Fragment or a funk tional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to item (c) or at least one antibody according to (d) (f) at least one compound according to one of claims 25 or 26 and / or (g ) at least one active ingredient which binds to at least one protein or a functional fragment or a functional variant thereof according to (a) for the manufacture of a medicament for the treatment of pain. Use according to claim 35 or 36, characterized in that the treatment is chronic, especially neuropathic or inflammatory pain. Use (c) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is able to specifically bind to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, d) at least one vector comprising a nucleic acid according to (a), and / or (c) at least one cell containing at least one nucleic acid according to (a) or at least one vector according to item (b) for gene therapy. Use according to claim 38, characterized in that it is in vivo or in vitro Gene therapy is acting. Use of (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 coded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins, (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1 , PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is able to specifically bind to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, c) at least one vector comprising a nucleic acid according to (b), (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), (e) at least one cell containing at least one protein or a functional one A fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to point (c) or at least one antibody according to (d) (f) at least one compound according to one of claims 25 or 26 and / or (g) at least one active ingredient which binds to at least one protein or a functional fragment or a function elle variant thereof according to (a) binds, for the diagnosis and / or for efficacy studies. Use of (a) at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1, PID12832121 / FLJ20420 and / or at least one protein comprising an amino acid sequence according to any one of 11 . 18 . 20 . 27 . 29 . 31 . 38 . 40 . 42 . 44 . 46 . 48 . 50 . 52 . 54 . 56 . 58 . 60 and / or at least one protein for which a nucleic acid comprising a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 coded, and / or at least one protein which is encoded by a nucleic acid which under stringent conditions with a nucleic acid comprising a nucleic acid sequence according to one of 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 or whose antisense nucleic acid hybridizes or a functional fragment or a functional variant of one of the aforementioned proteins, (b) at least one nucleic acid coding for at least one protein selected from the group consisting of KIAA0378, BAB31214 / Q9BU64, OPA1 / KIAA0567, CPG2 / KIAA1756, LETM1 , PID12832121 / FLJ20420 and PGRL or a functional fragment or a functional variant thereof and / or at least one nucleic acid comprising or consisting of a nucleic acid sequence according to one of the 12 . 19 . 21 . 28 . 30 . 32 . 39 . 41 . 43 . 45 . 47 . 49 . 51 . 53 . 55 . 57 . 59 . 61 and / or at least one nucleic acid, in particular an antisense nucleic acid or a PNA, which has a sequence which is capable of binding specifically to one of the abovementioned nucleic acids and / or a functional fragment or a functional variant of the at least one nucleic acid, (c) at least one vector comprising a nucleic acid according to (b), (d) at least one antibody against one of the proteins or a functional fragment or a functional variant thereof according to (a), and / or (e) at least one cell containing at least one A protein or a functional fragment or a functional variant thereof according to (a), at least one nucleic acid according to (b), at least one vector according to item (c) or at least one antibody according to (d) in a method for finding pain-regulating substances.