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WO2010116375A1 - Peptides isolés pour réguler l'apoptose - Google Patents

Peptides isolés pour réguler l'apoptose Download PDF

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Publication number
WO2010116375A1
WO2010116375A1 PCT/IL2010/000295 IL2010000295W WO2010116375A1 WO 2010116375 A1 WO2010116375 A1 WO 2010116375A1 IL 2010000295 W IL2010000295 W IL 2010000295W WO 2010116375 A1 WO2010116375 A1 WO 2010116375A1
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Prior art keywords
mtch2
peptide
mimp
apoptosis
isolated
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Inventor
Atan Gross
Yehudit Zaltsman
Assaf Friedler
Chen Katz
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Yeda Research and Development Co Ltd
Yissum Research Development Co of Hebrew University of Jerusalem
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Yeda Research and Development Co Ltd
Yissum Research Development Co of Hebrew University of Jerusalem
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention in some embodiments thereof, relates to isolated peptides which can increase or decrease apoptosis in a cell, and to methods of using same for treating disorders associate with abnormally low or high levels of apoptosis.
  • Programmed cell death, or apoptosis is essential for the development and maintenance of tissue homeostasis in multicellular organisms. Defects in apoptosis contribute to a variety of diseases, including cancer and neurodegenerative diseases. Thus, while oncogenesis and maintenance of the malignant phenotype of cancer cells involves blocking of death signaling, maintenance of the neurodegenerative phenotype of neuronal cells involves constitutive activation of death signaling. The most common abnormalities of cancer and neurodegenerative diseases are related to the mitochondrial apoptotic pathway, which involve the mitochondrial outer membrane permeabilization (MOMP).
  • MOMP mitochondrial outer membrane permeabilization
  • MOMP results in release of proteins from the intermembrane space to the cytosol (e.g., cytochrome c), leading to caspase protease activation and cell death.
  • BCL-2 family members are the major regulators of mitochondrial apoptosis, affecting the decision of "MOMP or no MOMP", which is translated into death or survival of the cell.
  • BAX and BAK are the pro-apoptotic effectors directly responsible for MOMP, and are antagonized by pro-survival proteins, including BCL-2, BCL-X L , and MCL-I.
  • BCL-2 and other pro-survival proteins may sequester BAX or BAK, making them unavailable for activation, or alternatively they may sequester BH3-only proteins that would otherwise bind and activate BAX and BAK.
  • BH3-mimetics and antagonize the functions of pro-survival BCL-2 family proteins have been identified.
  • BID has emerged as a key regulator of neuronal apoptosis, and several recent studies report the development of small-molecule BID inhibitors that provide a promising therapeutic strategy in neurodegenerative diseases (Becattini, B., et al., 2006;
  • the mitochondrial carrier homolog 2 (MTCH2/MIMP) [also called met-induced mitochondrial protein (MIMP)] is an evolutionary conserved protein, which carries six ⁇ -helixes that cross the outer mitochondrial membrane and interacts with the activated form of the BH3-only protein BID (tBID) in cells signaled to die by tumor necrosis factor-alpha (TNF ⁇ ) or FAS (Grinberg M., et al., 2005; Gross A, 2005).
  • TNF ⁇ tumor necrosis factor-alpha
  • FAS FAS
  • MTCH2/MIMP was shown to act as a tumor suppressor gene in mice (Leibowitz-Amit
  • PCT Patent Application IL2006/000021 discloses methods and pharmaceutical compositions for regulating apoptosis and treating pathologies associated with disregulated apoptosis using agents capable of modulating the expression of
  • an isolated peptide consisting of the amino acid sequence set forth by SEQ ID NO:106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 or 138.
  • an isolated peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs:106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 and 138, wherein the amino acid sequence is less than 60 amino acids in length and whereas the peptide decreases a level of apoptosis in a cell, with the proviso that the amino acid sequence is not the amino acid sequence set forth by SEQ ID NO:36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 or 69.
  • an isolated peptide comprising the amino acid sequence set forth by SEQ ID NOs:106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 and 138, wherein the amino acid sequence is less than 60 amino acids in length and whereas the
  • amino acid sequence is less than 60 in length and whereas the peptide increases a level of apoptosis in a cell.
  • an isolated peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs:lll, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110,
  • an isolated molecule comprising the isolated peptide of the invention, attached to an amino acid sequence which enhances penetration of the peptide into a cell.
  • an isolated molecule comprising the isolated peptide of the invention attached to an amino acid sequence which enhances penetration of the peptide into a cell.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence consisting of the amino acid sequence of the invention, or of the isolated molecule of the invention. According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence consisting of the amino acid sequence of the invention or of the isolated molecule of the invention.
  • nucleic acid construct comprising the isolated polynucleotide of the invention and a promoter for directing expression of the amino acid sequence in a host cell.
  • nucleic acid construct comprising the isolated polynucleotide of the invention and a promoter for directing expression of the amino acid sequence in a host cell.
  • a pharmaceutical composition comprising as an active ingredient the isolated peptide of the invention, the isolated molecule of the invention, the isolated polynucleotide of the invention or the nucleic acid construct of the invention.
  • a method of downregulating apoptosis in a cell comprising contacting the cell with the peptide of the invention, the isolated molecule of the invention, the isolated polynucleotide of the invention or the nucleic acid construct of the invention, thereby downregulating the apoptosis in the cell.
  • a method of upregulating apoptosis in a cell comprising contacting the cell with the peptide of the invention, the isolated molecule of the invention, the isolated polynucleotide of the invention or the nucleic acid construct of the invention, thereby upregulating the apoptosis in the cell.
  • a method of treating a pathology associated with abnormally high levels of apoptosis in a subject comprising administering to the subject a therapeutically effective amount of the peptide of the invention, the isolated molecule of the invention, the isolated polynucleotide of the invention or the nucleic acid construct of the invention, thereby treating the pathology associated with abnormally high levels of apoptosis in the subject.
  • a method of treating a pathology associated with abnormally low levels of apoptosis in a subject comprising administering to the subject a therapeutically effective amount of the peptide of the invention, the isolated molecule of the invention, the isolated polynucleotide of the invention or the nucleic acid construct of the invention, thereby treating the pathology associated with abnormally low levels of apoptosis in the subject.
  • the peptide is cyclic.
  • the pathology associated with abnormally high levels of apoptosis is a degenerative disorder.
  • the degenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and retinitis pigmentosa.
  • the pathology associated with abnormally high levels of apoptosis is human immunodeficiency virus (H ⁇ V)-induced acquired immunodeficiency syndrome (AIDS).
  • H ⁇ V human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • the pathology associated with abnormally low levels of apoptosis is selected from the group consisting of cancer, an autoimmune disorder, a bacterial infection, and a viral infection.
  • the amino acid sequence which enhances penetration of the peptide into a cell is set forth by SEQ ID NO: 131.
  • FIGs. IA-B are a diagram ( Figure IA) and a PCR analysis ( Figure IB) depicting the generation of MTCH2/MIMP knockout embryos.
  • Figure IA A diagram depicting the MTCH2/MIMP genomic locus, the targeting vector and the homologous recombinant, with the restriction enzyme sites (Ncol and Spel) and the position of external probes.
  • Figure IB - PCR analysis of wild-type, MTCH2/MIMP +/" heterozygote and MTCH2/MIMP "/" homozygote embryos. Two parallels are shown for each condition.
  • FIGs. 2A-E are histological analyses depicting morphological characteristics of the Mtch2 knock-out embryos.
  • Figures 2A and B are Sagittal sections of representative E7.5 wild-type ( Figure 2A) and MTCH2/MIMP 7" ( Figure 2B) embryos stained with hematoxylin and eosin.
  • the MTCH2/MIMP "/" embryos lack some of the typical structures of this stage: the chorion, amnion and ectoplacental cone are undetectable. Moreover, the extraembryonic region is unorganized and the mesoderm fails to migrate.
  • FIG. 2C a histogram depicting the number of cells in MTCH2/MIMP 7" E7.5 and wild-type E7.5 embryos. The cells of the ectodermal and mesodermal layers were counted in two wild-type and two knockout embryos. For each embryo three adjacent 4 ⁇ m sections were counted and the average value obtained. Note that MTCH2/MIMP "/" E7.5 embryos consist three times less cells than wild-type E7.5 embryos.
  • FIGS. 2D and E are images depicting Brachyury (T) mRNA expression patterns in E7.5 embryos using whole mount RNA in situ hybridization. Brachyury expression was detected by RNA in situ hybridization using an antisense dig-labeled ribo-probes.
  • Figure 2D - E7.5 wild-type embryos;
  • Figure 2E - E7.5 MTCH2/MIMP "/" embryos.
  • Brachyury mRNA is highly expressed in the primitive' streak at the posterior side of the embryonic (Em) region ( Figure 2D, marked by arrow).
  • FIG. 3B MTCH2/MIMP Western blot analysis.
  • the MTCH2/MIMP ⁇ ES cells were transfected with either an empty pcDNA3.1 vector (clone V42) or a pcDNA3.1 vector carrying MTCH2/MIMP (clone R5). Cells from two single stable clones (V42 and R5) were lysed, subjected to SDS-PAGE and Western blot analyzed using anti- MTCH2/MIMP antibodies.
  • the results demonstrate the presence of MTCH2/MIMP in cells rescued with a vector carrying the MTCH2/MIMP coding sequence.
  • FIG. 3C - tBID Western blot analysis.
  • V42 and R5 cells were infected with an adenovirus vector expressing the tBID coding sequence (Ad-HA-tBID), and mitochondria prepared from these cells were treated with the BS 3 cross-linker followed by Western blot analysis using anti-HA antibodies.
  • the results demonstrate that tBID cross-linked mitochondrial complex is generated in rescued cells (R) which express wild-type MTCH2/MIMP but not in cells devoid of MTCH2/MIMP (V cells).
  • FIGs. 3D-E are graphs depicting the effect of MTCH2/MIMP on mitochondrial membrane potential.
  • TMRE 10s
  • 5 mM succinate 50s
  • 2 mM ATP + CP/CPK 100s
  • 12 ⁇ g/ml oligomycin 150s
  • purified recombinant histidine-tagged murine tBID 300s
  • FCCP 5 ⁇ M 600s
  • actual raw data from representative experiments
  • TMRE fluorescence ex. 545, em.
  • V V42; Figure 3D; MTCH2/MIMP 7" ES cells rescued with an empty vector
  • R R5; Figure 3E; MTCH2/MIMP 'A ES cells rescued with a vector containing wild-type MTCH2/MIMP
  • Similar results were obtained with the two additional pairs of V and R clones.
  • the results demonstrate that the R cells (which include wild-type MTCH2/MIMP) are more sensitive than V cells (which are devoid of wild-type MTCH2/MIMP) to tBID-induced mitochondrial depolarization.
  • FIG. 3F is a Western blot analysis demonstrating cytochrome c (Cyt c) release as a function of wild-type MTCH2/MIMP.
  • Cyt c cytochrome c
  • FIG. 3G is a Western blot analysis depicting dimerization of BAX as a function of wild-type MTCH2/MIMP.
  • E the suspensions of the V42 and R5 clones were centrifuged and the pellet fractions were treated with the Sulfo-BSOCOES cross-linker and lysed. Equal amounts of protein (30 ⁇ g per lane) were subjected to SDS-PAGE, followed by Western blot analysis using anti-BAX Abs (651; gift from Stan Korsmeyer, DFCI, Boston, USA). * marks an intramolecular cross-linked product of activated BAX. The results shown in this Figure are representative of four independent experiments. Similar results were obtained with the two additional pairs of V and R clones. Note that BAX is homodimerized in the R cells at the low concentration of tBID.
  • FIGs. 4A-G demonstrate that conditional knockout of MTCH2/MIMP in MEFs reduces the sensitivity to tBID-induced apoptosis.
  • Figure 4A Generation of the MTCH2/MIMP conditional targeting vector. Indicated are loxP sites (black triangles), Frt sites (gray triangles), the neomycin (Neo) positive selection cassette, and the thymidine kinase (TK) negative selection cassette.
  • Figure 4B Western blot analysis of conditional deletion of MTCH2/MIMP in MEFs.
  • MTCH2/MIMP fllfl MEFs -/+ Cre- recombinase were lysed, and the mitochondria-enriched fractions were analyzed by Western blot for MTCH2/MIMP using an anti-MTCH2/MIMP antibody (Grinberg, M., Schwarz, M., Zaltsman, Y., Eini, T., Niv, H., Pietrokovski, S., and Gross, A. Mitochondrial Carrier Homolog 2 Is a Target of tBID in Cells Signaled To Die by Tumor Necrosis Factor Alpha. MoI Cell Biol, 25: 4579-4590, 2005). BAX was used as an internal standard.
  • Figure 4C - Western blot analysis was used as an internal standard.
  • MTCH2/MIMP fllfl MEFs were treated as in Figure 4B, infected with Ad-tBID and the mitochondria-enriched fractions were treated (+) or not (-) with the BSOCOES cross-linker followed by Western blot analysis.
  • CL cross-linker. * mark cross-reactive bands. Porin was used as an internal standard (bottom panel). The results show that tBID cross-linked complex is not generated in MrCH2/M/MF-deficient MEFs.
  • FIG 4D MTCH2/MIMP fl/fl (fl/fl; left panel) and MTCH2/MIMP fl/+ (fl/+; right panel) MEFs -/+ Cre-recombinase were infected with Ad-tBID and cell death was monitored by PI dye exclusion. Data are the mean ⁇ s.d. of three independent experiments. The results show that MTCH2IMIMP- deficient MEFs are less sensitive to Ad-tBID-induced apoptosis.
  • Figure 4E Fl/fl MEFs -/+ Cre-recombinase were infected with the indicated adenoviruses, and cell death was monitored as above.
  • FIGs. 5A-E demonstrate that conditional knockout of MTCH2/MIMP in MEFs hinders the recruitment of tBID to mitochondria.
  • FIG. 5A - fl/fl and fl/+ MEFs -/+ Cre-recombinase were treated with each of the indicated apoptotic stimuli for 14 hours: Fas (1 ng/ml) and cycloheximide (CHX; 1 ⁇ g/ml), Etop (100 ⁇ M), and Cis (33 ⁇ M). Cell death was monitored as above. Data are the mean ⁇ s.d. of three independent experiments, * F ⁇ 0.0005. The results show that MTCH2/M/MP-deficient MEFs are less sensitive to apoptosis induced by DNA-damaging reagents.
  • Figure 5B - Fl/fl MEFs -/+ Cre-recombinase were either infected with Ad-tBID, treated with Etop (100 ⁇ M; 8 hours), or treated with Fas (5 ng/ml) and cycloheximide (C ⁇ X; 1 ⁇ g/ml; 6 hours). Cells were then lysed, and the mitochondria-enriched fractions, the cytosolic fractions, and total cell lysates were Western blot analyzed with anti- ⁇ A (left top panel) or anti-BID (all other panels) Abs. * mark cross-reactive bands. Porin and actin were used as internal standards. The results show that deletion of MTCH2/MIMP hinders the recruitment of tBID to mitochondria.
  • Figure 5C - Fl/fl MEFs were treated as in Figure
  • FIGs. 6A-H demonstrate that MTCH2/MIMP deletion in the liver reduces the sensitivity of mice to Fas-induced hepatocellular apoptosis and hinders the recruitment of tBID to mitochondria.
  • Figure 6A Western blot analysis of MTCH2/MIMP in liver lysates demonstrates its absence in livers prepared from MTCH2 ) 1 MIMP* 1 / ⁇ ; AIb-Cr e (fl/ ⁇ ) mice. BCL-X L was used as an internal standard (bottom panel). * marks a cross reactive band.
  • Figure 6B Conditional knockout of MTCH2/MIMP in the liver significantly reduces the sensitivity of mice to Fas-induced hepatocellular apoptosis.
  • the Kaplan-Meier survival curves were compared using the long-rank test and were found statistically different froirreach other (P ⁇ 0.05).
  • Figures 6C-D Haematoxylin-eosin staining of paraffin-embedded liver sections from a fl/+ mouse ( Figure 6C) and a fl/ ⁇ mouse ( Figure 6D) 4 hours after anti-Fas antibody injection. Note the condensed and fragmented nuclei and the haemorrhage in the fl/+ liver. The bars represent 50 ⁇ m.
  • Figures 6E-F - fl/+ and fl/ ⁇ mice (four from each) were either left untreated (-) or injected with anti-Fas Abs for the indicated times.
  • FIG. 6G Liver mitochondria fractions prepared from the mice described in Figures 6E-F were lysed, and Western blot analyzed using anti-BID Abs.
  • Figure 6H Liver mitochondria fractions treated with trypsin, lysed, and Western blot analyzed using anti-BAX Abs. Porin was used in both blots as an internal standard. Note the significant decrease in tBID recruitment to mitochondria and BAX activation in fl/ ⁇ liver mitochondrial fractions in response to anti-Fas Ab.
  • FIGs. 7A-B are Western blot analyses demonstrating that MTCH2/MIMP deletion in the liver prevents the in vitro import of tBID.
  • Kinetics of HA-tBID import into mitochondria Figure 7A
  • Cyt c release Figure 7B
  • Cytsolic fractions of 293T cells expressing HA-tBID and depleted of Cyt c were incubated with purified, intact mitochondria isolated from mouse liver prepared from either fl/+ mice (top panels) or fl/ ⁇ mice (bottom panels).
  • mitochondria were separated from the soluble fraction by centrifugation, and both fractions were lysed and analyzed by Western blot with anti-HA (Figure 7A) or anti-Cyt c ( Figure 7B) Abs. Actin and porin were used as internal standards for the soluble/cytosolic and mitochondrial fractions, respectively.
  • FIGs. 8A-C are Western blot analyses of mouse liver mitochondria using antibodies directed against MTCH2/MIMP ( Figure 8A), AIF ( Figure 8B) and ANT ( Figure 8C).
  • Mouse liver mitochondria were either left untreated (-) or treated with a low (0.1 ⁇ g/ml; +) or a high concentration (1 ⁇ g/ml; ++) of proteinase K, lysed, size- fractionated by SDS-PAGE and analyzed by Western blot using anti-MTCH2/MIMP Abs (Figure 8A), anti-AIF (apoptosis inducing factor) Abs (Figure 8B), or anti-ANT (adenine nucleotide translocator) Abs ( Figure 8C).
  • FIGs. 8D-G are Western blot analyses of submitochondrial membrane vesicles using antibodies directed against cytochrome c oxidase subunit IV (Figure 8D), ANT ( Figure 8E), Tom20 ( Figure 8F) and MTCH2/MIMP ( Figure 8G).
  • Submitochondrial membrane vesicles were prepared from rat liver mitochondria, lyzed, size-fractionated by SDS-PAGE and analyzed by Western blot using anti-cytochrome c oxidase subunit IV (Cyt Oxi; Figure 8D) Abs, anti-ANT Abs (Figure 8E), anti-Tom20 Abs ( Figure 8F), and anti-MTCH2/MIMP Abs (Figure 8G).
  • OMM anti-cytochrome c oxidase subunit IV
  • Figure 8G anti-MTCH2/MIMP Abs
  • FIGs. 9A-B are immunoblot analyses depicting binding of recombinant tBID ( Figure 9A) or recombinant BID ( Figure 9B) to a MTCH2/MIMP - derived peptide array.
  • Cellulose-bound peptide array consisting of overlapping peptides derived from MTCH2/MIMP was screened by immunoblot experiments with recombinant tBID/BID proteins.
  • a dark spot represents binding of tBID/BID to a specific peptide as specified by the peptide reference number (rows E, F or G; columns 1-24).
  • the MTCH2/MIMP- derived peptides which bind the recombinant tBID/BID are provided in Table 4, Example 6 of the Examples section which follows.
  • FIG. 9C is a schematic presentation of the MTCH2/MIMP secondary structure with the position of the MTCH2/MIMP-derived peptides which bind BID or tBID according to the immunoblot results presented in Figures 9A-B.
  • the colors represent the degree of binding the BID/tBID proteins: Pink - peptides that do not bind BID or tBID; Dark green - Peptides which strongly bind tBID; Light green - peptides that bind tBID moderately; Light blue - peptides that bind weakly to tBID; Brown - peptides which bind full BID.
  • FIGs. 9D-F are schematic illustrations of the MTCH2/MIMP tertiary structure with the position of the tBID/full BID binding sites.
  • the tBID/full BID binding sites that were discovered in the peptide array screening ( Figures 9A-C, Table 4) are highlighted on the three-dimensional (3D) model structure that was recently generated [Schwarz, M., Andrade-Navarro, M. A., and Gross, A. (2007). Mitochondrial carriers and pores: key regulators of the mitochondrial apoptotic program? Apoptosis 12, 869- 876].
  • Figure 9D The tBID binding sites on the MTCH2/MIMP tertiary structure model.
  • the peptide that binds the tightest to tBID is colored using strong red and the other peptides are colored using light red;
  • Figure 9E The full length BID binding sites on the MTCH2/MIMP tertiary structure model.
  • the peptide that binds the tightest to full length BID is colored with green;
  • Figure 9F The tBID and full length BID binding sites on the MTCH2/MIMP tertiary structure.
  • FIG. 10 is a schematic illustration depicting a proposed model according to some embodiments of the invention for the regulation of tBID-induced MOMP by MTCH2/MIMP. Cleavage by caspase-8 generates tBID (light purple), which rapidly migrates to the membrane.
  • tBID induces a conformational change in BAX (pink; which includes N-terminus exposure) leading to its insertion into the membrane.
  • MTCH2/MIMP deep purple
  • tBID interacts with tBID and assists it in BAX activation.
  • the resulting activated BAX can oligomerize resulting in membrane permeabilization.
  • FIG. 11 depicts the amino acid sequence of mouse BID (SEQ ID NO:3) with the identified peptides following mass spectrometry results of cross-linking experiments using HA-tagged full length BID immunoprecipitated from transfected cells. Sequences of the seven peptides from the mouse BID protein that were identified in the MS analysis [highlighted with yellow, with a red box delineating each peptide; SEQ ID NOs:97, 100, 101, 102, 103, 104 and 105], and the five peptides that were not identified by mass-spec [non-highlighted, each peptide is delineated with a red box; SEQ ID NOs:96, 98, 99, 28, 29]. Marked in red is the cleavage site of BID to tBID, and in gray the potential sites of cross-linker binding in one of none-identified peptides.
  • FIGs. 12A-C are immunoblot analyses depicting binding of MTCH2/MIMP biotinylated peptide 240-290 (SEQ ID NO: 106) to a tBID/BID - derived peptide array.
  • Cellulose-bound peptide array consisting of overlapping peptides derived from tBID/BID was screened by immunoblot experiments with biotinylated peptide 240-290 (SEQ ID NO: 106) in different ionic strengths: 150 mM ( Figures 12A), 100 mM ( Figures 12B), 50 mM ( Figures 12C).
  • FIG. 13A is a schematic presentation of the tBID/BID secondary structure with the position of the tBID/BID -derived peptides which bind MTCH2/MIMP peptide 240- 290 (SEQ ID NO: 106) according to the immunoblot results presented in Figures 12A-C.
  • the colors represent the degree of binding the MTCH2/MIMP peptide 240-290: black - peptides that do not bind MTCH2/MIMP peptide 240-290 AA; Dark blue - Peptides which strongly bind MTCH2/MIMP peptide 240-290; Cyan - peptides that bind moderately-weakly to MTCH2/MIMP peptide 240-290.
  • FIG. 13B is a schematic illustration of the tBID/BID tertiary structure with the position of the MTCH2/MIMP peptide 240-290 (SEQ ID NO: 106) binding sites.
  • the tBID/full BID binding sites that were discovered in the peptide array screening ( Figures 12A-C,) are highlighted on the three-dimensional (3D) model structure PDB ID:2Bid.
  • the peptide that binds the tightest to MTCH2/MIMP peptide 240-290 is colored using strong blue and the other peptides are colored using cyan.
  • Table 8 Example 9 of the Examples section which i ⁇ llows.
  • "aa" (amino acid) residues marks the position of the peptides on the tBID/BID polypeptide sequence
  • FIGs. 14A-C are immunoblot analyses depicting binding of MTCH2/MIMP Biotinylated peptide 140-161 (SEQ ID NO: 107) to a tBID/BID - derived peptide array.
  • Cellulose-bound peptide array consisting of overlapping peptides derived from tBID/BID was screened by immunoblot experiments with biotinylated peptide 140-161 in different ionic strengths: 150 mM ( Figures 14A), 100 mM ( Figures 14B), 50 mM ( Figures 14C).
  • FIG. 15A is a schematic presentation of the tBID/BID secondary structure with the position of the tBID/BID -derived peptides which bind MTCH2/MIMP peptide 140- 161 (SEQ ID NO: 107) according to the immunoblot results presented in Figures 14A-C. All peptides that bound both the MTCH2 240-290 AA and the MTCH2 140-161 AA are highlighted as in Figure 13A, amino acid numbers indicated only the peptides that bound the MTCH2 140-161. Most peptides that bound the MTCH2 140-161, also bound the MTCH2 240-290, except for one additional peptide (tBID 62-76) which only bind to MTCH2 140-161 and is colored in pink.
  • FIG. 15B are schematic illustrations of the tBID/BID tertiary structure with the position of the MTCH2/MIMP peptide 140-161 (SEQ ID NO: 107) binding sites.
  • the tBID/full BID binding sites that were discovered in the peptide array screening ( Figures 14A-C,) are highlighted on the three-dimensional (3D) model structure PDB ID:2Bid.
  • the peptide that binds the tightest to MTCH2/MIMP peptide 140-161 is colored using strong blue and the other peptides are colored using cyan and one additional peptide that showed binding only to MTCH2 140-161 colored in pink.
  • "aa" (amino acid) residues marks the position of the peptides on the tBID/BID polypeptide sequence
  • FIG. 16 is a schematic illustration of the tBID/BID tertiary structure with the position of the MTCH2/MIMP peptides 140-161 (SEQ ID NO: 107) and 240-290 (SEQ ID NO: 106) binding sites.
  • the tBID/full BID binding sites that were discovered in the peptide array screening ( Figures 12-15,) are highlighted on the three-dimensional (3D) model structure PDB ID:2Bid.
  • the binding site of the peptides that bind the cleavage part of the protein (tBID) are colored in magenta, and the binding site represented by three peptides that bind the N-terminus of full length Bid are colored in blue.
  • aa amino acid residues marks the position of the peptides on the tBID/BID polypeptide sequence
  • FIGs. 17A-B demonstrate the conditional gene targeting of murine MTCH2/MIMP.
  • Figure 17A - A schematic illustration depicting the generation of the MTCH2/MIMP conditional targeting vector. Shown are the wild-type allele and the homologous recombinant product.
  • loxP sites which are excised by Cre recombinase; black triangles
  • Frt sites which are excised by FIp recombinase; gray triangles
  • the neomycin (Neo) positive selection cassette the thymidine kinase (TK) negative selection cassette
  • the restriction enzyme sites Xbal and Ncol and the position of the probes used to screen for MTCH2/MIMP +/ ⁇ ES clones by Southern blot.
  • Figure 17B Southern blot analysis demonstrating homologous recombination within the MTCH2/MIMP locus of one of the ES clones that was subsequently aggregated with tetraploid embryos.
  • FIGs. 18A-I demonstrate the conditional knockout of MTCH2/MIMP in MEFs.
  • Figure 18A A histogram depicting percentage of cell death in fl/fl MEFs following several apoptotic stimuli, fl/fl MEFs were either transduced or not with Cre- recombinase, and treated with each of the indicated apoptotic stimuli: TNF ⁇ (0.02 ng/ml) and actinomycin D (ActD; 2 ⁇ g/ml; 14 hours), Staurosporine (STS; 0.1 ⁇ M; 13 hours), and ultra-violet (UV; 10J/m 2 ; 8 hours). Cell death was monitored as in Figure 5A and the results are presented as mean ⁇ SD.
  • MTCH2/MIMP-def ⁇ cient MEFs are equally sensitive to several apoptotic stimuli.
  • Figure 18B Western blot analysis with ant-cleaved caspase antibodies. fl/fl MEFs were treated as above, lysed, and Western blot analyzed using anti-cleaved caspase-3 Abs. Actin was used as an internal standard (bottom panel). Note that deletion of MTCH2/MIMP reduces Ad- tBID- and Etop-induced caspase-3 cleavage/activation.
  • FIG 18C Western blot analysis with anti-Bax antibodies, fl/fl MEFs were either transduced or not with Cre- recombinase, and then treated with Ad-tBID, Etop or Fas as described in Figure 5C. Cells were then lysed, and the mitochondria-enriched fractions were treated with the BSOCOES cross-linker, followed by Western blot analysis. Note that deletion of MTCH2/MIMP hinders BAX dimerization.
  • Figures 18D-I - immuno-fluorescence analyses using cytochrome c antibodies, fl/fl MEFs were either transduced ( Figures 18E, 18G and 181) or not ( Figures 18D, 18F and 18H) with Cre-recombinase, and then plated on coverslips in a 12 well plate (80,000 cells/well). Cells were then exposed to the indicated death stimuli [Ad-tBID (10 MOI), Figures 18D-E; Etop (100 ⁇ M), Figures 18F-G; Fas (1 ng/ml) plus cycloheximide (CHX; 1 ⁇ g/ml), Figures 18H and I, for 14, 8 and 7 hours, respectively].
  • Ad-tBID (10 MOI)
  • Figures 18D-E Etop (100 ⁇ M)
  • Figures 18F-G Fas (1 ng/ml) plus cycloheximide (CHX; 1 ⁇ g/ml)
  • Figures 18H and I for
  • FIGs. 19A-C are PCR analyses depicting genotyping of mouse tails of progeny that were generated from crosses between MTCH2/MIMP +//> ;Mb-C ⁇ e and MTCHUMIMP* 11 * 1 mice.
  • the liver specific knockout mouse No.
  • the control heterozygote littermate (No. #69) carries a wild-type allele (+), a floxed allele (fl), and the Alb-Cre gene.
  • mice numbers refer to: #65: MTCH2/MIMP +/ ⁇ ; Alb-Cre; #66: MTCH2/MIMP fl/ ⁇ ; Alb-Cre; #67: MTCH2/MIMP fl/ ⁇ ; #68: MTCH2/MIMP +/ ⁇ ; #69: MTCH2/MIMP fl/+ ; Alb-Cre.
  • FIG. 20 is a histogram depicting the level of serum liver enzymes aspartate aminotransferase (AST) and alanineaminotransferase (ALT) in fl/+ and fl/ ⁇ mice. Serum was collected after over night fast. Data are presented as mean ⁇ SEM.
  • the present invention in some embodiments thereof, relates to isolated peptides which can increase or decrease apoptosis in a cell and to methods of using same for treating cancer or neurodegenerative diseases.
  • the present inventors have uncovered novel MTCH2/MIMP-derived peptides which bind BID and/or tBID and which can decrease the level of apoptosis in cells and treat pathologies associated with excessive apoptosis such as neurodegenerative diseases.
  • the present inventors have uncovered BID- and tBID-derived peptides which can increase the level of apoptosis in cells and treat pathologies associated with abnormally low levels of apoptosis such as cancer.
  • MTCH2/MIMP is required for embryonic development and that a complete absence of MTCH2/MIMP is embryonic lethal (Example 1, Table 3, Figures IA-B and
  • MTCH2/MIMP is a positive regulator of tBID-induced BAK/BAX activation and mitochondrial outer membrane permeabilization (MOMP) (Example 2, Figures 3 A-G).
  • conditional knockout of MTCH2/MIMP in MEFs reduces the sensitivity to tBID-induced apoptosis (Example 3, Figures 4A-G, 17A-B), and hinders the recruitment of tBID to mitochondria (Example 4, Figures 5A-E; 18A-I).
  • MTCH2/MIMP deletion in the liver reduces the sensitivity of mice to fas-induced hepatocellular apoptosis and hinders the recruitment of tBID to mitochondria ( Figures 6A-H, 19-20).
  • Example 6 MTCH2/MIMP deletion in the liver prevents the in vitro import of tBID ( Figures 7A- B). In addition, it was found that MTCH2/MIMP is exposed on the surface of mitochondria (Example 5, Figures 8A-G).
  • the present inventors identified MTCH2/MIMP-derived peptides which bind tBID or BID (SEQ ID NOs: 106, 107, 16- 26 for human MTCH2/MIMP-derived peptides; Example 8, Tables 4 and 5, Figures 9A- D) and which can reduce apoptosis in cells, and novel BID- and tBID-derived peptides which bind MTCH2/MIMP (SEQ ID NOs:30, 31, 32, 115, 111, 114, 112, 113, 108, 109 or 110 for human BID- and tBID-derived peptides; Examples 9, 10 and 11, Tables 6, 7, 8, and 9, Figures 11, 12, 13, 14, 15 and 16) and which can increase apoptosis in cells.
  • an isolated peptide comprising the amino acid sequence set forth by SEQ ID NO:111, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117, wherein the amino acid sequence is less than 60 in length and whereas the peptide increases a level of apoptosis in a cell.
  • the isolated peptide is less than 60 amino acids in length, e.g., 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 or 40 amino acids, e.g., less than 40 amino acids in length, e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31 or 30 amino acids, e.g., less than 30 amino acids in length, e.g., 29, 28, 27, 26, 25, 24, 24, 22, 21 or 20 amino acids, e.g., less than 20 amino acids in length, e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 amino acids, or less.
  • the isolated peptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 111, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117.
  • the isolated peptide which comprises the amino acid sequence selected from the group consisting of SEQ ID NOsrlll, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117 is capable of increasing the level of apoptosis in a cell.
  • an isolated peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 and 138, wherein the amino acid sequence is less than 60 in length and whereas the peptide decreases a level of apoptosis in a cell, with the proviso that the amino acid sequence is not the amino acid sequence set forth by TYALDSGVSTMNEMKSYSQA (SEQ ID NO:36), YALDSGVSTMNEMKSYSQAV (SEQ ID NO:37), ALDSGVSTMNEMKSYSQAVT (SEQ ID NO:38), LDSGVSTMNEMKSYSQAVTG (SEQ ID NO:39), DSGVSTMNEMKSYSQAVTGF (SEQ ID NO:40), SGVSTMNEMKSYSQAVTGFF (SEQ ID NO:41), YPFVLVSNLMAVNNCGLAGG (SEQ ID NOs:36), YALDSGVSTMNEMKSYS
  • the isolated peptide consists of the amino acids sequence set forth by SEQ ID NO:106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 or 138.
  • the isolated peptide which comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 and 138 is capable of decreasing the level of apoptosis in a cell.
  • isolated refers to at least partially separated from the natural environment e.g., the human body.
  • the term "isolated” refers to a soluble molecule.
  • peptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • mimetics refers to molecular structures, which serve as substitutes for the peptide of the invention in performing the biological activity (Morgan et al. (1989) Ann. Reports Med. Chem. 24:243-252 for a review of peptide mimetics) such as peptoids and oligopeptoids, which are peptides or oligomers of N- substituted amino acids [Simon et al. (1972) Proc. Natl. Acad. Sci. USA 89:9367-9371].
  • Peptide mimetics may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of the peptide. Further included as peptide mimetics are peptide libraries, which are collections of peptides designed to be of a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto. Methods of producing peptide mimetics are described hereinbelow.
  • Peptide bonds (-C0-NH-) within the peptide may be substituted, for example, by
  • Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (NoI), ring-methylated derivatives of
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxyzine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non- conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with the invention.
  • Table 1
  • the peptides of the invention can be utilized in a linear or a cyclic form. According to some embodiments of the invention, the peptide is a cyclic peptide. Peptides with a cyclic backbone have been described in the art for increasing their ability to penetrate a cell-of-interest (see e.g., Hariton-Gazal E, et al., 2005).
  • the peptides of the invention may include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • peptides of the invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis.
  • solid phase peptide synthesis a summary of the many techniques may be found in J. M. Stewart and J. D. Young,
  • Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.
  • these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain.
  • amino acids or suitably protected amino acids Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
  • the protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage.
  • the protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final peptide compound.
  • a preferred method of preparing the peptide compounds of the invention involves solid phase peptide synthesis. Large scale peptide synthesis is described by Andersson Biopolymers 2000;55(3):227-50.
  • Combinatorial chemical, antibody or peptide libraries may be used to screen a plurality of peptides or mimetics thereof.
  • peptides can be generated using recombinant DNA techniques.
  • an isolated polynucleotide sequence encoding the amino acid sequence of the isolated peptide of the invention e.g., SEQ ID NOs:106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 or 138
  • a nucleic acid construct suitable for expression in a host cell e.g., SEQ ID NOs:106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 or 138
  • Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner
  • the nucleic acid construct of the invention may also include an enhancer, a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal, a 5 1 LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof; a signal sequence for secretion of the peptide from a host cell; additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide;
  • IRS internal ribosome entry site
  • mammalian expression vectors include, but are not limited to, pcDNA3, ⁇ cDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pB V- IMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149: 51-60).
  • AcMNPV Autographa californica nucleopolyhedrovirus
  • Recombinant viral vectors are useful for in vivo expression since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • the expression construct of the present invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide.
  • a fusion protein or a cleavable fusion protein comprising the protein of the invention (the gene product of the polynucleotide-of-interest) and a heterologous protein can be engineered.
  • Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein.
  • the protein of the invention can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].
  • an appropriate enzyme or agent that disrupts the cleavage site e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].
  • prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention.
  • host-expression systems include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence.
  • Mammalian expression systems can also be used to express the polypeptides of the present invention.
  • polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the peptide of the invention may be further conjugated to an amino acid sequence, which facilitates penetration of the peptide into a cell or further into a subcellular organelle such as the nucleus, nucleoli, mitochondria and the like.
  • the peptide can be conjugated to a known sequence such as the P18 (LSTAADMQGVVTDGMASG; SEQ ID NO:70) or P28
  • LSTAADMQGVVTDGMASGLDKDYLKPDD azurin-derived peptides
  • the peptide can be conjugated to the cationic cell-penetrating peptides Tat [CRKKRRQRRR (SEQ ID NO:72)], oligoarginine [r9; CRRRRRRRRR (SEQ ID NO:73)] or oligolysine [k9; CKKKKKKKKK (SEQ ID NO:74)] described in Patel LN, et al., MoI. Pharm.
  • the peptide can be conjugated to the nuclear localization signal (NLS) [KKKRKV (SEQ ID NO:75)] or to the basic TAT peptide [GRKKRRQRRR (SEQ ID NO:76)] described in Peitz, M., et al., 2002, "Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes".
  • NLS nuclear localization signal
  • GRKKRRQRRR SEQ ID NO:76
  • the peptide can be conjugated to PenetratinTM 1 Peptide [QBiogene Molecular Biology; e.g., Activated Penetratin 1 Peptide (Cat. No. PENA0500; Biotinylated Activated Penetratin 1 Peptide (Cat. No. PENB0500)].
  • Conjugation of a cell-penetrating amino acid sequence to the isolated peptide of the invention can be performed using methods known in the art.
  • the cell- penetrating amino acid sequence can be conjugated via, for example, a disulfide bridge to a d-isoform cysteine (c) present at the N-terminal of the isolated peptide of the invention.
  • the cell-penetrating amino acid sequence can be recombinantly synthesized along with the peptide of the invention from a nucleic acid construct encoding both sequences.
  • the peptide of the invention may be also conjugated to a non-proteinaceous moiety, which increases the stability (e.g., against protease activities) and/or solubility (e.g., within a biological fluid such as blood, digestive fluid) of the peptide while preserving the biological activity and prolonging the half -life of the peptide.
  • the non-proteinaceous moiety can be a polymer or a co-polymer (synthetic or natural) such as polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), divinyl ether and maleic anhydride copolymer (DIVEMA; see for example, Kaneda Y, et al., 1997, Biochem. Biophys. Res. Commun. 239: 160-5) and poly(styrene comaleic anhydride) (SMA; see for example, Mu Y, et al., 1999, Biochem Biophys Res Commun. 255: 75- 9
  • Bioconjugation is advantageous particularly in cases of therapeutically useful proteins which exhibit short half -life and rapid clearance from the blood.
  • the increased half -lives of bioconjugated proteins in the plasma results from increased size of protein conjugates (which limits their glomerular filtration) and decreased proteolysis due to polymer steric hindrance.
  • the more polymer chains attached per peptide the greater the extension of half-life.
  • measures are taken not to reduce the specific activity of the isolated peptide of the invention.
  • Bioconjugation with PEG ⁇ i.e., PEGylation can be performed using PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG 2 -NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG- orthopyridyl-disulfide, carbonyldimidazol-activated PEGs, PEG-thiol, PEG-maleimide.
  • PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG 2 -NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG
  • PEG derivatives are commercially available at various molecular weights [See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle, Ala.)]. If desired, many of the above derivatives are available in a monofunctional monomethoxyPEG (mPEG) form.
  • mPEG monomethoxyPEG
  • the PEG added to the amino acid sequence of the peptide of the invention should range from a molecular weight (MW) of several hundred Daltons to about 100 kDa (e.g., between 3-30 kDa). Larger MW PEG may be used, but may result in some loss of yield of PEGylated peptides.
  • PEG purity of larger PEG molecules should be also watched, as it may be difficult to obtain larger MW PEG of purity as high as that obtainable for lower MW PEG. It is preferable to use PEG of at least 85 % purity, and more preferably of at least 90 % purity, 95 % purity, or higher. PEGylation of molecules is further discussed in, e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Dunn and Ottenbrite, eds., Polymeric Drugs and Drug Delivery Systems, American Chemical Society, Washington, D.C. (1991).
  • PEG can be attached to a chosen position in the amino acid sequence by site-specific mutagenesis as long as the activity of the conjugate is retained (e.g., decrease of apoptosis).
  • a Cysteine residue can be a target for PEGylation.
  • Computational analysis may be effected to select a preferred position for mutagenesis without compromising the activity.
  • activated PEG such as PEG-maleimide, PEG- vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide
  • Methods of preparing activated PEG molecules are known in the arts.
  • PEG-VS can be prepared under argon by reacting a dichloromethane (DCM) solution of the PEG-OH with NaH and then with di-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2 gram PEG/mL DCM).
  • DCM dichloromethane
  • PEG-AC is made under argon by reacting a DCM solution of the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM).
  • acryloyl chloride and triethylamine molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM.
  • Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.
  • cysteine residues While conjugation to cysteine residues is one convenient method by which the peptide of the invention can be PEGylated, other residues can also be used if desired.
  • acetic anhydride can be used to react with NH 2 and SH groups, but not COOH, S--S, or -SCH 3 groups
  • hydrogen peroxide can be used to react with — SH and -SCH 3 groups, but not NH 2 .
  • Reactions can be conducted under conditions appropriate for conjugation to a desired residue in the peptide employing chemistries exploiting well-established reactivities.
  • the terminal COOH-bearing PVP is synthesized from N-vinyl-2-pyrrolidone by radical polymerization in dimethyl formamide with the aid of 4,4'-azobis-(4-cyanovaleric acid) as a radical initiator, and 3-mercaptopropionic acid as a chain transfer agent.
  • Resultant PVPs with an average molecular weight of Mr 6,000 can be separated and purified by high-performance liquid chromatography and the terminal COOH group of synthetic PVP is activated by the N-hydroxysuccinimide/dicyclohexyl carbodiimide method.
  • the isolated peptide of the invention is reacted with a 60-fold molar excess of activated PVP and the reaction is stopped with amino caploic acid (5-fold molar excess against activated PVP), essentially as described in Haruhiko Kamada, et al., 2000, Cancer Research 60: 6416-6420, which is fully incorporated herein by reference.
  • Resultant conjugated peptide molecules e.g., PEGylated or PVP-conjugated CCR2 are separated, purified and qualified using e.g., high-performance liquid chromatography (HPLC).
  • purified conjugated molecules of this aspect of the invention may be further qualified using e.g., in vitro assays in which the level of apoptosis in a cell is tested (as is further described hereinbelow) in the presence or absence of the peptide-conjugates of the invention.
  • in vitro assays in which the level of apoptosis in a cell is tested (as is further described hereinbelow) in the presence or absence of the peptide-conjugates of the invention.
  • the BID derived peptide comprises the amino acid sequence set forth by SEQ ID NOrIlJ., 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117, wherein the amino acid sequence is less than 60 in length and whereas the peptide increases a level of apoptosis in a cell.
  • the isolated peptide is set forth by SEQ ID NOs.lll, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117.
  • the phrase "increase of apoptosis” refers to an increase in the rate of apoptosis in a cell(s) and/or an increase in the number of cells undergoing apoptosis in a tissue or subject.
  • apoptosis refers to a programmed cell death machinery whereby the cell executes a "cell suicide” program. Apoptosis plays a crucial role in ensuring the normal development and maintenance of cells, organs, and tissues and involves in a number of physiological events such as embryogenesis, regulation of the immune system, and homeostasis.
  • apoptosis can be in response to diverse signals such as stimulation by growth factors (e.g., TNF ⁇ and Fas), limb and neural development, neurodegenerative diseases, radiotherapy and chemotherapy as well as environmental conditions.
  • Apoptotic processes are usually characterized by uncoupling of mitochondrial oxidation, decreased levels of nicotinamide adenine dinucleotide phosphate [NAD(P)H], release of cytochrome c, activation of caspases, DNA fragmentation and externalization of phosphatidylserine (a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer) to the outer leaflet of the plasma membrane (described in length in the preceding background section).
  • growth factors e.g., TNF ⁇ and Fas
  • Apoptotic processes are usually characterized by uncoupling of mitochondrial oxidation, decreased levels of nicotinamide adenine dinucleotide phosphate [NAD(P)H], release of cytochrome c,
  • a method of upregulating apoptosis in a cell comprising contacting the cell with the isolated peptide, isolated molecule and/or the isolated polynucleotide or nucleic acid construct encoding same of the invention, thereby upregulating the apoptosis in the cell.
  • the cell can be any cell such as an embryonic or adult cell, a stem cell, a progenitor cell, a fetal or adult blood cell, a bone marrow cell, a neuronal cell, a cardiac cell, a bone cell, a muscle cell, and the like.
  • contacting the cells with the peptide can be performed under in vitro or in vivo conditions.
  • the cells are contacted with the peptide e.g., by adding the peptide to cells derived from a subject (e.g., a primary cell culture, a cell line) or to a biological sample comprising same (e.g., a fluid, liquid which comprises the cells) such that the peptide is in direct contact with the cells.
  • the cells of the subject are incubated with the peptide.
  • the conditions used for incubating the cells are selected for a time period/concentration of cells/concentration of peptide/ratio between cells and peptide and the like which enable the peptide to induce cellular changes, such as reduction in the rate of apoptosis.
  • the effect of the isolated peptides or molecules comprising same of the invention on apoptosis can be determined, for example, using functional assays, such the Ethidium homodimer-1 staining (Invitrogen-Molecular Probes), the Tunnel assay (Roche, Basel, Switzerland), the Live/dead viability/cytotoxicity two-color fluorescence assay (Molecular Probes, Inc., L-3224, Eugene, OR, USA), FACS analysis [using molecules capable of specifically binding cells undergoing apoptosis, such as propidium iodide and Annexin V], and those of skills in the art are capable of assessing such levels in order to determine the standards of normal levels.
  • functional assays such as the Ethidium homodimer-1 staining (Invitrogen-Molecular Probes), the Tunnel assay (Roche, Basel, Switzerland), the Live/dead viability/cytotoxicity two-color fluorescence assay (Molecular Probes, Inc., L-3224, Eugene, OR, USA), FACS
  • the agents of the invention which increase the level of apoptosis in a cell can be used to treat a pathology associated with abnormally low levels of apoptosis in a subject, by administering to the subject a therapeutically effective amount of the isolated peptide (e.g., SEQ ED NO:111, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117), the isolated molecule comprising same and/or the isolated polynucleotide or the nucleic acid construct encoding same.
  • the isolated peptide e.g., SEQ ED NO:111, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 or 117
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term "subject” refers to an animal subject e.g., a mammal, e.g., a human being at any age who suffers from or is at risk of developing the pathology.
  • pathology refers to any deviation from the normal structure and/or function of a particular cell, cell type, group of cells, tissue or organ leading to a disease, a disorder, a syndrome or an abnormal condition.
  • abnormally lowJevels of apoptosis relates to any pathology which is caused by, characterized by or associated with a rate and/or level of apoptosis which is below (Le., abnormally low) the level present in normal or unaffected cells of the same type or developmental stage.
  • Normal or unaffected cells can be obtained from a subject who is devoid of the pathology, e.g., from a subject who does not suffer from the pathology or its symptoms, and/or is not predisposed to have the pathology.
  • Pathologies which are caused by, characterized by or associated with abnormally low levels of apoptosis and which can be treated using the agents of the present invention include, but are not limited to, cancer, autoimmune disorders associated with low level of apoptosis of auto-reactive lymphocytes, bacterial infection associated with downregulation of apoptosis in host cell [e.g., bacterial infections caused by Chlamydia sp., Neisseria sp., Salmonella enterica, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Rickettsia rickettsii, Wolbachia Neutrophils, Bartonella sp., Helicobacter pylori, Porphyromonas gingivalis, Listeria monocytogenes, Shigella flexneri, Legionella pneumophila, Mycoplasma fermentans, Brucella suis, Escherichia coli Kl, and Coxiella burnetii (for further details
  • the peptide according to some embodiments of the invention decreases a level of apoptosis in a cell.
  • a method of downregulating apoptosis in a cell the method is effected by contacting the cell with the isolated peptide, the isolated molecule comprising same and/or the isolated polynucleotide or nucleic acid construct encoding same of the invention, thereby downregulating the apoptosis in the cell.
  • the peptide which is capable of decreasing the level of apoptosis in cells is selected from the group consisting of SEQ ID NOs: 106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25 and 26.
  • the human derived peptide MTCH2/MIMP 240-290 (SEQ ID NO: 136), the mouse derived peptide MTCH2 240-292 (SEQ ID NO: 137) and/or the human derived peptide MTCH2/MIMP 2402-92 (SEQ ID NO: 138) can be also used to reduce the level of apoptosis in a cell.
  • the phrase "decrease of apoptosis" refers to a decrease in the rate of apoptosis in a cell(s) and/or a decrease in the number of cells undergoing apoptosis in a tissue or subject.
  • the teachings of the invention can be used to treat a pathology caused by, associated with or characterized by abnormally high levels of apoptosis in a subject.
  • abnormally high levels of apoptosis relates to any pathology which is caused by, characterized by or associated with a rate and/or level of apoptosis which is above (i.e., abnormally high) the level present in normal or unaffected cells of the same type or developmental stage.
  • Pathologies which are caused by, associated with or characterized with abnormally high levels of apoptosis and which can be treated using the isolated peptide, the isolated molecule and/or the isolated polynucleotide or nucleic acid construct encoding same of the invention include, but are not limited to, degenerative disorders such as neurological disorders [e.g., a neurodegenerative disorder such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and retinitis pigmentosa], atherosclerosis (Mercer J., et al., 2007, Mutation Research 621: 75-86), or pathologies associated with viral infections such as central nervous system (CNS) diseases [e.g., human immunodeficiency virus (H ⁇ V)-induced associated dementia (Li, W., Galey D et al., 2005, Neurotox res.
  • degenerative disorders such as neurological disorders [e.g., a neurodegenerative disorder such as Alzheimer's disease, Parkinson's disease,
  • herpes simplex virus -induced encephalitis Perkins D., Gyure K., et al., 2003, J. Neurovirol. 9:101-111
  • cytomegalovirus-induced encephalitis DeBiasi R.L., et al., 2002, J. Infect. Dis. 186: 1547-1557
  • heart diseases e.g., active and chronic myocarditid (Alter P., et al., 2001, Cardiovasc. Patholog. 10:229-234)
  • liver diseases e.g., hepatitis B or C virus associated liver injury (Bantel H., et al., 2003, Cell Death Differ. 10 (suppl.
  • the isolated peptide, isolated molecule comprising same and/or isolated polynucleotide or nucleic acid construct encoding same of the invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the isolated peptide, isolated molecule comprising same and/or the isolated polynucleotide or the nucleic acid construct encoding same of the invention accountable for the biological effect.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • CNS central nervous system
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the blood brain barrier (BBB)] in an attempt to exploit one of the endogenous transport pathways of the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent e.g., conjugation of water-soluble agents to lipid or cholesterol carriers
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., the isolated peptide, the isolated molecule comprising same and/or the isolated polynucleotide or the nucleic acid construct encoding same) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a neurodegenerative disease or cancer) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the isolated peptide and/or the isolated molecule comprising same can be administered to the individual [e.g., systemically (e.g., intravenous, intramuscularly) or locally to the target tissue or organ, e.g., brain, using for example an implanted pump].
  • systemically e.g., intravenous, intramuscularly
  • target tissue or organ e.g., brain
  • the isolated polynucleotide or the nucleic acid construct encoding the peptide of the invention can be targeted to the brain using liposomal and viral vectors as described in de Lima MC, et al., 2005, Curr Drug Targets CNS Neurol Disord. 4(4):453-65, which is fully incorporated herein by reference, and/or using a neuron-specific promoter such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473- 5477].
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the isolated peptides or molecules comprising same of the invention can be qualified for their ability to upregulate or downregulate apoptosis as needed. This can be done, for example, using functional assays, such as by monitoring the effect of the -peptide/molecule of the invention on apoptosis in cells.
  • the level of apoptosis in cells and tissues can be determined using various methods such as the Ethidium homodimer- 1 staining (Invitrogen-Molecular Probes), the Tunnel assay (Roche, Basel, Switzerland), the Live/dead viability/cytotoxicity two-color fluorescence assay (Molecular Probes, Inc., L-3224, Eugene, OR, USA), FACS analysis [using molecules capable of specifically binding cells undergoing apoptosis, such as propidium iodide and Annexin V], and those of skills in the art are capable of assessing such levels in order to determine the standards of normal levels.
  • Ethidium homodimer- 1 staining Invitrogen-Molecular Probes
  • the Tunnel assay (Roche, Basel, Switzerland)
  • the Live/dead viability/cytotoxicity two-color fluorescence assay Molecular Probes, Inc., L-3224, Eugene, OR, USA
  • FACS analysis using molecules capable of specifically binding cells undergoing apoptosis, such as
  • the teachings of the invention can be used to screen for peptides which can upregulate or downregulate apoptosis.
  • peptides can be qualified for their ability to increase or decrease the binding of MTCH2 with BID or the binding of MTCH2 with tBID.
  • the peptides described hereinabove can be used to generate antibodies which bind the MTCH2/MIMP or the BID polypeptides in a biological sample of a subject. Such antibodies can be used for both diagnostic and therapeutic methods.
  • the biological sample can be any sample which contains proteins of the subject.
  • the biological sample can include cells or cell content such as a body fluid [e.g., whole blood, white blood cells, peripheral blood mononuclear cells (PBMCs), serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva or milk] or a tissue biopsy.
  • a body fluid e.g., whole blood, white blood cells, peripheral blood mononuclear cells (PBMCs), serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva or milk
  • a tissue biopsy e.g., whole blood, white blood cells, peripheral blood mononuclear cells (PBMCs), serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva or milk
  • a tissue biopsy e.g., whole blood, white
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab")2, and Fv that are capable of binding to macrophages.
  • These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab 1 fragments are obtained per antibody molecule; (3) (Fab")2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab”)2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain
  • the isolated peptide of the invention can be conjugated to an immunogenic moiety such as keyhole limpet haemocyanin (KLH) (Imject Maleimide-activated mcKLH from Pierce, according to the manufacturer's protocol) and be further subcutaneously injected in several places on the back of an experimental animal (e.g., rabbit). Conventially, following a predetermined period, such as two weeks, a booster prepared with incomplete Freund's adjuvant (Sigma) is injected. Following 2-3 weeks, blood is collected from the animal, red blood cells are clotted and removed and the serum is centrifuged (e.g., for 30 minutes at 1500 g). The resulting supernatant contains the antibodies.
  • KLH keyhole limpet haemocyanin
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Natl Acad. Sci. USA 69:2659-62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between" a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number "to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the targeting vector was designed to delete 2.4 Kb, encompassing exons 1-3 of MTCH2/MIMP [GenBank Accession No. AF176009 (SEQ ID NO:34; gi:5815346); encoding GenBank Accession No. AAD52647; gi:5815347].
  • the targeting vector was constructed using PCR on ES genomic DNA (Galli-Taliadoros, L. A., J. D. Sedgwick, S. A. Wood, and H. Korner. 1995. Gene knock-out technology: a methodological overview for the interested novice.
  • the linearized targeting vector was introduced into Rl ES cells (derived from 129/ola mice) by electroporation and ⁇ 1,000 neomycin resistant clones were picked. The individual clones described above were screened for homologous recombination by Southern blot analysis.
  • Southern blot analysis for the MTCH2/MIMP knockout mice -
  • a 5' probe and a 3' probe were constructed.
  • the 5' probe which is located -500 bp upstream to the SH, was prepared by a PCR reaction with the following primers: 5'-TATTTGGATCCTCCTAACCAGTTTAGATGGTTGC-S' (SEQ ID NO:82; forward primer) and
  • the 3' probe which is located ⁇ 250 bp downstream to the LH, was prepared by a PCR reaction with the following primers:
  • the probes were synthesized using PCR and labeled with ⁇ - 32 P-dCTP (3,000 ci/mmol, Amersham) using the random primer DNA labeling kit (Biological Industries Beit-Haemek). The hybridization was performed in rapid-hybridization buffer (Amersham) according to manufacturer's protocol and specific binding was analyzed by autoradiography.
  • MTCH2 IMIMP knockout mice Two homologous recombinant Rl clones were identified, aggregated with tetraploid embryos and implanted into separate white-coated ICR foster mother mice. The first generation of black-coated mice were born, bred again to white ICR mice, to obtain the second generation of MTCH2/MIMP + ⁇ animals. Intercross of MTCH2/MIMP +/" animals resulted in offspring homozygous for the Mtch2/Mimp knock out (MTCH2/MIMP 7 ). Timed pregnancies, isolation of embryos, and PCR analysis - Timed pregnancies were conducted with MTCH2/MIMP +/ ⁇ mice.
  • E0.5 embryonic development day 0.5
  • Pregnant females were sacrificed at different time points of gestation, and embryos were dissected from maternal tissue.
  • Uteri from E6.5, E7.5, E8.5 and E9.5 pregnancies were isolated in ice-cold phosphate-buffered saline. Decidua were separated, embryos dissected out under a binocular, and pictures were taken.
  • DNA was prepared using the Epicentre MasterPure purification kit and than analysed by PCR.
  • the yolk sac was separated and lysed using the REDExtract-N-Amp Tissue PCR Kit (Sigma), and the sets of primers that were used for genotyping of pups were employed.
  • the whole uterus was fixed in 4% paraformaldehyde for 48 hrs at RT. Sections were cut from paraffin blocks and stained with hematoxylin and eosin (H&E).
  • ES cells of MTCH2 /MIMP knockout mice Pregnant females from MTCH2/MIMP +/" intercrosses were sacrificed at E3.5, and blastocysts were collected by flushing the uteri (Hogan, B., R. Beddington, F. Constantini, and E. Lacy. 1994. Manipulating the mouse embryo. Cold Spring Harbor Laboratory Press, Plainview, N. Y). Blastocysts were cultured individually in 96-well plates in DMEM (Gibco) supplemented with 20 % fetal bovine serum (FBS) (Gibco), 1 mM NaPyruvate,
  • MTCH2/MIMP "7" ES cells were transfected with either an empty pcDNA3.1 vector or a pcDNA3.1 vector carrying MTCH2/MIMP using Lipofectamine 2000 (Invitrogen). The cells were then cultured under selective conditions (medium containing 2 mg/ml hygromycin), and surviving clones were expanded and used as stable clones for the experiments described.
  • This vector consists of all the elements needed for conditional gene targeting: two loxP sites, a PGKneo cassette (which provides neomycin resistant) flanked by two Frt sites (that enable the excision of the PGKneo cassette upon FIp recombinase expression), an ampicylin resistant cassette, and a thymidine kinase (TK) cassette (which serves as a negative selection against random, non-homologous integration of the construct to the genome).
  • the targeting vector was constructed by PCR using 129/SVJ genomic DNA. First, each of the PCR products was ligated into the pGEM T-Easy vector (Promega), and colonies that carried the expected insert size were taken for sequencing.
  • the "best" colony (the one with the sequence that most resembled the sequence that appears in the data base) was chosen and suspected mutations where corrected by site directed mutagenesis.
  • the second step was to clone the isolated PCR products into the pRapidflirt vector.
  • the long homology (LH) arm which consists of 7 Kb upstream to exon-1 was ligated into Xhol and Fsel sites in the pRapidflirt vector down stream to the TK cassette.
  • the forward primer contained both the cloning Xhol site and an AfIII site that was later used to linearize the targeting vector, and the reverse primer contained the Fsel site.
  • the primers used for the LH-PCR reaction were: S'-CCGCTCGAGCTTAAGTGACCATATGACCTTTCCAT-S' (SEQ ID NO:86; forward) and 5'-CGACGTGGCCGGCCAAAGTTTGATGGTTGTnTC-S 1 (SEQ ID NO:87; reverse).
  • a 2.9 Kb DNA fragment which consists of the 5'-UTR, the first three exons of MTCH2/MIMP and a small portion of the third intron (named Ex; Figure 4A was ligated into Sail and Sbfl sites of the pRapidflirt vector between the two loxP sites (the first loxP site is located downstream to the 5'-UTR of MTCH2/MIMP and the other one is located upstream to the Frt site of the PGKneo cassette).
  • the primers used for the Ex-PCR reaction were: 5'-
  • ACGCGTCGACTCTAGAACGTCGTCAAAGCCTGAAAG-3' (SEQ ID NO:88; forward) which contained the Sail cloning site and Xbal site [that is further used in the Southern blot screen for identifying MTCH2/MIMP flox/neo positive embryonic stem (ES) cells], and S'-CAGAGAACCTGCAGGAGAGATGCCATGCCAGAGTTA-S' (SEQ ID NO.89; reverse) which contains the Sbfl site.
  • the short homologues (SH) arm which contains 2 Kb of the third intron was ligated into the Notl and CIaI restriction sites of the pRapidflirt vector.
  • the primers used for the SH-PCR reaction were: 5'- AAGGAAAAAAGCGGCCGCTTCTCTTGAAAGACATTTTC-3' (SEQ ID NO:90; forward) and 5'- CCCATCGATTrCTTTGCCTTTTTCTCTTTC-3' (SEQ ID NO:91; reverse). Subsequently, the complete targeting vector was subjected to sequence analysis, and the -18.8 Kb linearized vector was introduced into Rl ES cells by electroporation.
  • Southern blot analysis for the MTCH2/MIMP conditional knockout mice were screened by Southern blot analysis using two probes that were designed to detect wild-type and conditional alleles.
  • the 5 '-probe which is located 20 bp upstream to the LH arm was prepared by a PCR reaction with the following primers: 5'-TGAGCATGGAAGCAATGAAG-3 1 (SEQ ID NO:92; forward) and 5'- TGTTCTGGTTTGCTCTGTGG -3' (SEQ ID NO:93; reverse).
  • the 3' probe which is located 280 bp downstream of the SH arm, was prepared by a PCR reaction with the following primers: 5'-AACCCGTCTTGCTTCTACCAG-3 1 (SEQ ID NO:94; forward) and 5'-GGTGGGCACTACCATACCTG-S' (SEQ ID NO:95; reverse).
  • the PCR products were cloned into the pGEM T-Easy vector. Genomic DNA was digested with either Xbal or Ncol restriction enzymes, separated on a 0.8 % agarose gel and transferred to Hybond-N + membrane (Amersham) in 0.1 N NaOH.
  • the probes were labeled with ⁇ - 32 P-dCTP (3000 ci/mmole, Amersham) using the random primer DNA labeling kit (Biological Industries Beit-Haemek).
  • the hybridization was preformed in rapid-hybridization buffer (Amersham) according to the manufacturer's instructions and the radioactive signal of the specific binding of the labeled probe was analyzed by exposure to a high sensitive film (Kodak). ⁇ 750 neomycin resistant clones were picked, 400 individual ES clones were screened for homologous recombination by Southern blot analysis and ten clones showed the correct homologous recombination event.
  • MTCH2 IMIMP conditional knockout mice Two homologous recombinant Rl clones were identified, aggregated with tetraploid embryos and implanted into separate white-coated ICR foster mother mice. The first generation of black-coated mice were born, bred again to white ICR mice, to obtain the second generation of MTCH2/MIMP flox/+ (MTCH2/MIMP fl/+ ) animals.
  • mice with a pure 129 inbred background both the ES cells and the tetraploid embryos that were used to create the chimeras have the 129 background
  • confirmed chimeras with germ-line transmission were mated to wild-type mice from the 129/SVJ line.
  • the PGKneo cassette was excised by crossing the MTCH2/MIMP n/+ mouse to a general FIp deleter mouse that expresses the FIp recombinase in all tissues (e.g., the Rosa-Flp mouse).
  • MEFs with Cre-recombinase - MTCH2/MIMP fl/fl and MTCH2/MIMP fl/+ primary MEFs were prepared from 11- to 13- day-old embryos, and maintained in ISCOVE' s medium containing 10 % fetal bovine serum.
  • SV40 transformation of primary MEFs was performed by transfecting cells with the SV40 whole genome using Lipofectamine 2000 (Invitrogen). Stable clones were collected 14-to-18 days post transfection. All the studies with MEFs described in the paper were performed with SV40-immortalized MEFs.
  • HTNC His-TAT-NLS- Cre
  • PI propidium iodide
  • the mitochondrial pellet was resuspended in SEM (250 mM sucrose, 10 mM MOPS/KOH, 2.5 mM EDTA) together with 0.1 or 1 ⁇ g/ml proteinase K, and incubated at 4 0 C for 20 minute.
  • the reaction was stopped with 1 mM PMSF and the mitochondria were centrifuged at 10,000 X g for 10 minutes, resuspended in HIM buffer (200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM HEPES, pH 7.5) containing 1 mM PMSF, and again recentrifuged at 10,000 X g for 10 minutes.
  • the pellet was resuspended and incubated in a hyposmotic potassium phosphate buffer (swelling medium, 10 mM KH 2 PO 4 , pH 7.4) for 30 minutes on ice.
  • a shrinking medium 32 % sucrose, 30 % glycerol, 10 mM MgCl 2 ) was added to the suspension (33 % volume).
  • the mitochondrial membranes were disrupted by ultrasonication (4 x 1 minute of irradiation, 1-minute break between each run). The resulted material was spun down at 12,000 X g for 10 minutes.
  • the supernatant contains a mixture of mitochondrial membrane vesicles and was used as a reference for the further purified membranes.
  • a discontinuous sucrose gradient was used (from bottom to top, 70, 45.6, 34.2, and 26 % sucrose steps, 200,000 X g for 240 minutes).
  • the OMM was concentrated in the interface between the 26 and 34.2 % steps, and the IMM was collected between 45.6 and 70 %.
  • the protein concentration was determined in each fraction.
  • Livers from four female mice were used to prepare each OMM sample. Livers were excised and mitochondria were prepared as was previously described [Grinberg, M. et al. Mitochondrial Carrier Homolog 2 Is a Target of tBID in Cells Signaled To Die by Tumor Necrosis Factor Alpha. MoI Cell Biol 25, 4579-90 (2005)] with several modifications. The final mitochondria-enriched pellet was gently resuspended in 50 ml of MB buffer (210 mM Mannitol, 70 mM Sucrose, 10 mM Hepes, 1 mM EDTA, pH 7.5) and purified on a discontinuous Nycodenz (Sigma) gradient according to [Da Cruz, S. et al.
  • the purified mitochondria pellet was resuspended (5 mg protein/ml) and incubated in hyposmotic potassium phosphate buffer (swelling medium; 10 mM KH 2 PO 4 pH 7.4 for 30 min on ice while stirring). Subsequently, a shrinking medium (32% sucrose, 30% glycerol, 10 mM MgCl 2 ) was added to the suspension (33% volume). After 30 min of shrinking the mitochondrial membranes were disrupted by ultrasonication 4 x 20 seconds with a 1 min break between runs. The resultant material was spun down at 12,000 x g for 10 min.
  • hyposmotic potassium phosphate buffer swelling medium
  • a shrinking medium 30% sucrose, 30% glycerol, 10 mM MgCl 2
  • the supernatant containing a mixture of mitochondrial membrane vesicles was carefully layered on a discontinuous sucrose gradient (from bottom to top 70, 45.6, 34.2, and 26% sucrose, 2 ml each in SW41 rotor test tubes), and centrifuged for 12-to- 16 hrs (200,000 x g, 4° C).
  • the OMM was concentrated in the interface between the 26 and 34.2% steps and further washed in 70 ml MB buffer and centrifuged (141,000 x g, 4 hrs). The resulting pellet was resuspended in MB buffer and immediately frozen in liquid N2 for Western blot analysis.
  • Caspase-3 activity assay in hepatocytes were performed as previously described with some modifications [Sarig, R. et al. BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts. J Biol Chem 278, 10707-15 (2003)]. Livers were minced, washed in ice-cold PBS, and homogenized in lysis buffer (20 mM HEPES pH 7.3, 5 mM EGTA, 5 mM EDTA, 10 ⁇ M digitonin, 2 mM DTT) using a 2-ml Wheaton Dounce glass homogenizer and a glass "B"-type pestle.
  • the lysates were clarified by centrifugation and the supernatants were used for the assays. Enzymatic reactions were carried out in lysis buffer containing 20 ⁇ g of protein and 50 ⁇ M acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC; Alexis) to assess caspase-3 activity. Each sample was divided into three parts: One of them included in addition to the extract and substrate, 50 ⁇ M zVAD-fmk (BioMol) to inhibit caspase activity and two replicates that included extract and substrate, without inhibitor.
  • DEVD-AMC acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin
  • reaction mixtures were incubated at 37 0 C and fluorescent AMC formation was measured at excitation 380 nm and emission 460 nm using a Wallac Victor2 1420 Multilabel Counter (PerkinElmer). Specific activity was calculated for each sample as the mean of the duplicate sample minus the value obtained for the sample containing zVAD-fmk. Knockdown of MTCH2/MIMP in U2OS cells - Human MTCH2/MIMP was knockdown using siRNA On-TargetPlus smart pools (Dharmacon).
  • Preparation of recombinant adenoviruses and infection of MEFs - tBID and GFP recombinant adenoviruses were prepared as was previously described [Sarig, R. et al. BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts. J Biol Chem 278, 10707-15 (2003), which is hereby incorporated by reference in its entirety].
  • MTCH2/MIMP, BAX, Noxa, and BCL-2 recombinant adenoviruses were prepared as was previously described for preparing tBID recombinant adenoviruses [Sarig, R. et al. 2003].
  • Bim recombinant adenoviruses were prepared as was previously described for preparing Nbk recombinant adenoviruses [Gillissen, B. et al. Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway. Embo J 22, 3580-90 (2003), which is hereby incorporated by reference in its entirety].
  • Viruses were grown using 293T-TR cells. Virus preparations were made from freeze/thaw lysis of the cells, and virus titers were done on 293T-TR cells. In experiments, cells were generally seeded at 70-80% confluence. Cells were infected with an MOI (multiplicity of infection) of 10. Efficiency of infection was determined using the GFP recombinant adenovirus and was in the range of 70-to-90%.
  • MOI multiplicity of infection
  • TMRE fluorescence recordings were carried out at ex. 545 nm, em. 580 run. At the end of the recordings, the suspensions of the cells were centrifuged and the pellets were separated from the supernantants. Supernatants were used to analyze Cyt c release, and pellets were used to analyze BAK and BAX dimerization.
  • tBID a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14:2060-71).
  • proteins were size-fractionated by SDS-PAGE, then transferred to PVDF membranes (Immobilon-P, Bio-Rad), and Western blots were developed by use of the enhanced chemiluminescence reagent (NEN).
  • Antibodies used for Western blot included anti- MTCH2/MIMP Ab (Grinberg, M., et al., 2005. MoI Cell Biol 25:4579-90), anti-BID Ab (Grinberg, M., et al., 2005.
  • mice generation of MTCH2 IMIMP liver-specific knockout mice -
  • the existing Cre systems provide variable efficiencies which are rather weak in most cases.
  • the present inventors generated mice in which one of the MTCH2/MIMP alleles was fully deleted and the other one knocked out only in the organ of target. These mice were generated by first mating the MTCH2/MIMP fll mice with mice bearing Pgk-Cre, a general deleter transgene [Lallemand, Y., Luria, V., Haffner-Krausz, R. & Lonai, P.
  • PGK-Cre transgene Maternally expressed PGK-Cre transgene as a tool " fo ⁇ early and uniform activation of the Cre site-specific recombinase. Transgenic Res 7, 105-12 (1998)] to create MTCH2IMIMP* IA mice. These mice were then mated with mice bearing Alb-Cre, a transgene for Cre-recombinase under control of the liver albumin promoter [Postic, C. et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274, 305-15 (1999)].
  • MtchZ' V zc ⁇ o ⁇ and Mtch2 '/' Mtch2 lines were permeabilized using digitonin followed by the addition of recombinant tBID (a concentrations of 1 nM and 40 nM). Cytochrome c release was induced by recombinant tBID. At the end of the experiment, the suspension was centrifuged and the cell pellet was separated from the supernantant.
  • yeast mitochondria and in vitro import assays Wild-type yeast mitochondria were isolated from cultures grown at 30 0 C to an OD 6 oo of 2 in rich lactate medium (1 % yeast extract, 2 % tryptone, 0.05 % dextrose, and 2 % lactic acid, 3.4 mM CaCl 2 2H 2 O, 8.5 mM NaCl, 2.95 mM MgCl 2 6H 2 O, 7.35 mM KH 2 PO 4 , and 18.7 mM NH 4 Cl). Mitochondria were isolated as previously described (Claypool, S. M., et al., 2006.
  • the reticulocyte lysate containing the radiolabeled precursor was incubated at 30 0 C with isolated mitochondria in import buffer (1 mg/ml bovine serum albumin, 0.6 M sorbitol, 150 mM KCl, 10 mM MgCl 2 , 2.5 mM EDTA, 2 mM ATP, 2 mM NADH, and 20 mM Hepes-KOH, pH 7.4). Where indicated, the potential across the mitochondrial inner membrane was dissipated using 1 ⁇ M valinomycin and 25 ⁇ M FCCP. Unimported radiolabeled precursor was removed by treatment with 10 ⁇ g/ml trypsin for 30 minutes on ice.
  • import buffer 1 mg/ml bovine serum albumin, 0.6 M sorbitol, 150 mM KCl, 10 mM MgCl 2 , 2.5 mM EDTA, 2 mM ATP, 2 mM NADH, and 20 mM Hepes-KOH, pH 7.
  • Mitochondria were collected by spinning at 8,000 X g for 5 minutes at 4 °C and resuspended in 0.1 M sodium carbonate at various pHs or a total fraction in Thorner buffer (10 % glycerol, 8 M urea, 5 % SDS, 40 mM Tris, pH 6.8, 4 mg/ml bromophenol blue, and 5 % ⁇ -mercaptoethanol).
  • the mitochondria were pelleted at 20,000 X g for 15 minutes at 4 °C.
  • the pellet containing the membrane fraction was resuspended in Thorner buffer, and the supernatants were precipitated with 20 % trichloroacetic acid for 30 minutes on ice.
  • the supernatants were then spun at 20,000 X g for 15 minutes at 4 °C. These pellets were then resuspended in Thorner buffer.
  • Bacterial expression of MTCH2/MIMP and transport assays The coding sequences for human and murine MTCH2/MIMP (accession numbers AY380792.1 and NP_062732.1, respectively) were amplified from human and murine liver cDNA by PCR.
  • the oligonucleotide primers corresponded to the extremities of the coding sequences with additional Ndel and EcoRI restriction sites.
  • the amplified products were cloned into the pMW7 expression vector (Fiermonte, G., et al., 1998, J Biol Chem 273:24754-9) and sequenced.
  • the human and the murine MTCH2/MIMP were overexpressed as inclusion bodies in the cytosol of E. coli C0214(DE3) (Fiermonte, G., et al., 1998, J Biol Chem 273:24754-9). Control cultures with the empty vector were processed in parallel.
  • Inclusion bodies were purified on a sucrose density gradient [Fiermonte, G., et al., 1993, Biochem J 294 ( Pt l):293-9] and analyzed by SDS-PAGE. The proteins were solubilized in 1.8 % sarkosyl (w/v). Small residues were removed by centrifugation (258,000 X g, 1 hour).
  • Solubilized proteins were reconstituted into liposomes followed by four different procedures, specific for the functional reconstitution of the bovine oxoglutarate (OGC) [Fiermonte, G., et al., 1993, Biochem J 294 (Pt l):293-9], the human aspartate/glutamate (isoform 2, AGC2) (Palmieri, L., et al., 2001, Embo J 20:5060-9), the ATP-Mg/Pi (isoform 1, APCl) (Fiermonte, G., et al., 2004, J Biol Chem 279:30722-30), and the yeast NAD + (isoform 1, Ndtlp) (Todisco, S., et al., 2006, J Biol Chem 281:1524-31) recombinant mitochondrial carriers, used as positive controls.
  • OPC bovine oxoglutarate
  • Extraliposomal labeled substrate was removed from quenched samples on Sephadex G-75, and eluted radioactivity was measured (Palmieri, F., et al., 1995, Methods Enzymol 260:349-69). Transport activities were calculated from the experimental values minus the controls.
  • Immunofluorescence and imaging For imaging, cells on coverslips were fixed with 3 % formaldehyde in PBS in permeabilized with 0.2 % Triton X-100/PBS. Cells were immunostained with anti-cytochrome c 6H2.B4 monoclonal antibodies (PharMingen) followed by Cy3-conjugated goat anti-IgG (Jackson ImmunoResearch). Nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; 10 ⁇ g/ml). Images were collected on an Olympus 1X70 microscope, equipped with Deltavision imaging system, using a 40 x PLAN-APO 1.42NA objective. Images were processed by constrained iterative deconvolution on softWoRxTM software (Applied Precision).
  • HA-tBID cross-linked complex 100 x 10 cm plates of 293T cells were transiently transfected with pcDNA3-HA-mBID (GenBank Accession No. MMU75506; SEQ ID NO:33 (mRNA of mouse BID); gi: 1669513). Eighteen hours post-transfection, cells were harvested and subcellularly fractionated by differential centrifugation, as described above. The mitochondria-enriched heavy membrane fractions were treated with sulfo-BSOCOES (Sulfo-Bis[2-(sulfosuccinimidooxy- carbonyloxy)ethyl]sulfone; (Pierce)) at a final concentration of 10 mM.
  • sulfo-BSOCOES Sulfo-Bis[2-(sulfosuccinimidooxy- carbonyloxy)ethyl]sulfone
  • the cross-linker was quenched by the addition of 1 M Tris-HCl (pH 7.5) to a final concentration of 20 mM.
  • the membrane fraction was separated from the soluble fraction by centrifugation, and lysed in Laemli sample buffer without reducing agents. The resulting lysate was diluted in binding buffer [20 mM Tris (pH 7.5), 0.1 M NaCl, 0.1 M EDTA] to reach a final concentration of 0.2 % SDS.
  • the diluted lysate was incubated for 16 hours with 5 mg anti-HA mAbs coupled to agarose beads (Roche), followed by extensive washing of the beads with binding buffer containing 0.05 % Tween-20.
  • the material that remained bound to the beads was eluted by incubation with 1 ml (1 mg/ml) HA peptide at 37 °C for 15 minutes. Elufion was repeated twice more, and the three elutents were pooled and concentrated, using a Centricon tube with a 3K cutoff (Amicon). The concentrated material was loaded onto a single lane, separated by SDS-PAGE, and then stained with Coomassie blue.
  • the gel pieces were then incubated overnight at 37 °C and the resulting peptides were recovered with 60 % acetonitrile with 0.1 % trifluoroacetate.
  • the tryptic peptides were resolved by reverse- phase chromatography on 0.1 X 300-mm fused silica capillaries (100 micrometer ID, J&W) filled with porous R2 (Perspective).
  • the peptides were then eluted using a 80- min linear gradient of 5 to 95 % acetonitrile with 0.1 % acetic acid in water, at a flow rate of 1 ⁇ l/minute.
  • the liquid from the column was electrosprayed into an ion-trap mass spectrometer (LCQ, Finnegan, San Jose). '
  • Mass spectrometry was performed in the positive ion mode, utilizing a repetitively full MS scan, followed by collision-induced dissociation (CID) of the most dominant ion selected from the first MS scan.
  • CID collision-induced dissociation
  • the mass spectrometry data was compared to simulated proteolysis and CID of the proteins in the NR-NCBI database, using Sequest software (J. Eng, University of Washington, and J. Yates, Finnegan, San Jose).
  • the amino terminal of the protein was sequenced on Peptide Sequencer 494A (Perkin Elmer) according to the manufacturer's instructions.
  • Statistical analysis - Data are presented as the mean ⁇ s.d. Student's unpaired two-tailed Mest was performed using Microsoft Excel statistical analysis functions. Differences were considered statistically significant at P ⁇ 0.05.
  • the Kaplan-Meier survival curves were compared using the long-rank test (PASW Statistics 17.0 software).
  • MTCHHMIMP Loss of MTCHHMIMP results in embryonic lethality -
  • the MTCH2/MIMP gene spans -23 Kb on chromosome 2 and consists of 13 exons.
  • the wild-type MTCH2/MIMP allele, the targeting construct, and the targeted allele are illustrated in Figure IA. In the targeted allele, the first three exons were replaced with the neomycin resistant cassette, thereby creating an MTCH2/MIMP null allele.
  • mice Distribution of genotypes during embryonic development oj MTCH2/MIMP knock out mice
  • MTCH2/MIMP +/ were intercrossed (timed pregnancies) and the frequency of each genotype in all embryos was determined during embryonic development from embryonic day 6.5 (E6.5) through embryonic day 10.5 (E10.5).
  • +/+ homozygote wild-type embryos carrying two wild- type alleles i.e., two copies of the normal, functional MTCH2/MIMP
  • N/D genotype not determined.
  • E7.5 MtchHMimp '1' embryos are significantly smaller then the wild-type embryos and lack structures that are typical to this stage -
  • the present inventors have focused on E7.5 embryos. Pregnant females were sacrificed at E7.5, and the whole uterus was subjected to histological sectioning.
  • Figures 2A-B show sections of a representative wild-type ( Figure 2A) and MTCH2/MIMP 7" ( Figure 2B) E7.5 embryo stained with hematoxylin and eosin.
  • Several major morphological differences were noted between the wild-type and the MTCH2/MIMP-knock-out embryos.
  • the wild-type embryo has an oval and elongated morphology, whereas the MTCH2/MIMP "/" embryo is significantly smaller in size and rounder in shape. Counting the number of cells in the ectodermal and mesodermal layers in two wild-type and two knockout embryos indicated that the knockout embryos have three times less cells than the wild-type embryos ( Figure 2C); 2) The wild-type embryo has well defined extraembryonic (ExEm) and embryonic (Em) regions as expected (Kaufman, M. 1999. The anatomical basis of mouse development. Academic Press, San Diego, CA), whereas in the knockout embryo there is no formal organization of the ExEm region (only one large cavity can be detected; Figures 2A-B).
  • ExEm extraembryonic
  • Em embryonic
  • MTCH2/MIMP deficiency reduces the sensitivity to tBID-induced MOMP - Mtch2/Mimp " ⁇ ES cells generated from E3.5 blastocytes were confirmed to lack the Mtch2 protein by Western blot analysis using anti-MTCH2/MIMP antibodies ( Figure 3A).
  • the Mtch2/Mimp " ⁇ ES cells were then transfected with either an empty vector or a vector carrying MTCH2/MIMP fused to a Myc-His (MH) tag, and several stable lines carrying either the empty vector (V clones) or MTCH2/MIMP-MH (Rescue or R clones) were generated ( Figure 3B; note that the levels of MTCH2/MIMP-MH in the R clones were significantly lower than the levels of the endogenous MTCH2/MIMP in wild-type ES cells).
  • tBID forms a ⁇ 45 kDa cross-linkable complex in mitochondria prepared from apoptotic cells
  • MTCH2/MIMP is the protein that associates with tBID in this complex
  • mouse embryonic fibroblasts (MEFs) were isolated from homozygous Mtch2/Mimp fl/fl embryos and transduced with purified Cre-recombinase, leading to efficient deletion of MTCH2/MIMP in vitro ( Figure 4B).
  • Cross-linking experiments confirmed that tBID forms the ⁇ 45kD tBID-MTCH2/MIMP cross-linkable complex in MTCH2/MIMP fllfl cells but not in the same cells transduced with purified Cre-recombinase (Fig. 4C).
  • homozygous MTCH2/MIMP fl/fl and hetrozygous MTCH2/MIMP fll+ MEFs were transduced with Cre- recombinase, infected with HA-tagged tBID [Ad-tBID; Sarig, R. et al. BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts. J Biol Chem 278, 10707- 15 (2003)] and cell death was monitored.
  • MTCH2/MIMP fl/fl (fl/fl) cells transduced with Cre-recombinase were significantly less sensitive to Ad- tBID ( Figure 4D).
  • reintroduction of MTCH2/MIMP into fl/fl MEFs transduced with Cre-recombinase fully restored susceptibility to tBID-induced cell death ( Figure 4E).
  • MTCH2/MIMP plays an important role in tBID-induced cell death.
  • MTCH2/MIMP is conserved in different mammalian species, MTCH2/MIMP was knocked down in human U2OS cells, and this knockdown resulted in a -40% reduction in Ad-tBID-induced cell death ( Figure 3G).
  • MTCH2/MIMP plays a role in the pro-apoptotic action of tBID also in human cells.
  • the present inventors also found that deletion of MTCH2/MIMP significantly reduced the formation of BAX homodimers in cells treated with all three stimuli (Figure 18C). Based on these results the present inventors anticipated that MTCH2/MIMP deletion also reduces MOMP, and indeed it was found that fl/fl MEFs transduced with Cre-recombinase showed significantly less Cyt c release following treatment with all three stimuli ( Figure 5D and Figures 18D-I). Thus, deletion of MTCH2/MIMP hinders tBID recruitment to mitochondria, resulting in less BAX activation and MOMP.
  • fl/fl MEFs are type I cells (i.e., cells in which the mitochondrial pathway does not determine the time course of death receptor-induced apoptosis).
  • the present inventors found that fl/fl MEFs infected with recombinant adenoviruses carrying the BCL-2 vector were protected from Etop but not from Fas-induced cell death (Figure 5E).
  • BID is a critical substrate in vivo for signaling by death-receptor agonists, which mediates a mitochondrial amplification loop essential for the apoptosis of hepatocytes
  • Yin, X. M. et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400, 886-91 (1999)].
  • MTCH2/MIMP is a critical component of the tBID-death pathway in vivo
  • the present inventors generated MTCH2/MIMP liver-specific knockout mice using the Alb-Cre transgene (Figure 19).
  • mice were injected with anti-Fas antibodies, and the mice were sacrificed at three time points post injection (the fl/ ⁇ mice were sacrificed at a 2 hour delay due to their delayed death).
  • the eight livers including livers from two non- injected mice) were lysed, and the cytosolic and mitochondrial fractions were taken for analysis. Analysis of the cytosolic fractions of the fl/+ livers indicated that caspase-8, BID, and caspase-3 have been cleaved/activated ( Figure 6E, top, middle, and bottom panels, respectively).
  • MTCH2/MIMP IS EXPOSED ON THE SURFACE OF MITOCHONDRIA AND IS LOCALIZED TO THE OMM FRACTION
  • MTCH2/MIMP is related to members of the mitochondrial carrier (MC) protein family and therefore is likely to be localized to the inner mitochondrial membrane (IMM).
  • IMM inner mitochondrial membrane
  • apoptosis inducing factor [AIF, localized to the intermembrane space (IMS)]
  • ANT adenine nucleotide translocator
  • Submitochondrial membrane vesicles were prepared from rat liver mitochondria by sonication and separated by centrifugation through a discontinuous sucrose gradient, as was previously described (Bathori, G., et al., 2006, J Biol Chem 281:17347-58).
  • the high-density fractions are enriched in inner membrane vesicles as demonstrated by the presence of the inner membrane proteins cytochrome c oxidase and ANT, whereas the low-density fractions are enriched in outer membrane vesicles as demonstrated by the presence of the outer membrane protein Tom20 ( Figures 8D-G).
  • MTCH2/MIMP is most prominent in the low-density fractions, indicating that it is enriched at the outer membrane.
  • Figures 68-G show that MTCH2/MIMP is exposed on the surface of mitochondria and is localized to the OMM fraction.
  • MTCH2/MIMP interacts with tBID in mitochondria of apoptotic cells, and that this protein is found as part of a large complex in native mitochondria and that tBID and BAX are recruited to this complex and that BCL-X L inhibits this recruitment, suggesting that MTCH2/MIMP plays a positive role in regulating tBID/BAX-induced apoptosis (Grinberg, M., et al., 2005, MoI
  • V and R cells showed similar mitochondrial targeting of tBID but the V cells showed less tBID-induced dimerization of BAX and BAK (at the low concentrations of tBID; Figure 3G), suggesting that MTCH2/MIMP is involved in regulating tBID-induced activation of BAX and BAK, thus MTCH2/MIMP is positively regulating tBID-induced MOMP.
  • MrCH2/M/MP-deficient MEFs showed a significant reduction in tBID recruitment to mitochondria following Ad-tBID, Etop and Fas treatment ( Figure 5A-E). MrCH2/M/MP-deficient MEFs also hindered BAX activation and Cyt c release following all three stimuli.
  • MTC ⁇ 2/MIMP acts as a tBID receptor-like protein in the OMM to facilitate tBID recruitment, resulting in accelerated/effective BAX activation and MOMP.
  • MTCH2/MIMP acts at the very early stages of MOMP by facilitating the recruitment of tBID, the initiator of this process.
  • the studies in mice demonstrate that MTCH2/MIMP is an indispensable player in the tBID-death pathway required for effective hepatocellular apoptosis.
  • a peptide array containing 37 overlapping peptides derived from the sequence of the full-length mouse Mtch2/Mimp (SEQ ID NO:1) was designed. Peptide length was between 10-30 residues. Peptides were designed based on the predicted secondary structure of the Mtch2/Mimp protein, and recombinant tBID or BID were screened for binding the peptide array using the procedure described in (Hayouka, Z., et al., 2007).
  • tBID bound peptides are derived from two sites that are distant from each other according to their linear position on the Mtch2 sequence but are in spatial proximity according to the Mtch2/Mimp model ( Figures 9A-D and Table 4, hereinbelow).
  • the first site consists of helix 6, between residues 140-161 (SEQ ID NO:5), and the second site consists of the whole C-terminal part of the protein, between residues 240-300 (SEQ ID NOs:8-12).
  • This second site is represented by several peptides that bind Mtch2/Mimp with different strengths, with peptide F17 (SEQ ID NO:8; located at position 240-254 of the mouse Mtch2 protein set forth by SEQ ID NO:1) binding the tightest to tBID ( Figure 9D, colored strong red).
  • Table 4 Peptides from the mouse MTCH2/MIMP (SEQ ID NO:1; GenBank Accession No. GenBank accession number AAD52647; gi:5815347) which bind the mouse tBID or BID proteins according to the peptide-array immunoblot ( Figures 9A-B).
  • Table 5 Peptides from the human MTCH2/MIMP (SEQ ID NO:2; GenBank Accession No. NP_055157; gi:7657347) which are predicted to bind the human tBID or BID proteins based on the homology to the mouse Mtch2/Mimp.
  • tBID-derived peptides that bind to Mtch2 have employed a cross-linking strategy followed by mass spectroscopy analysis, as follows. Experimental Results Identification of BID-derived peptides that bind Mtch2 - The present inventors have previously demonstrated that HA-tBID is capable of forming a 45 kDa complex in 293T cells that represents a complex with MTCH2/MIMP.
  • the HA-BID complex (which included mouse BID) was purified and the peptides that are involved in the interaction with Mtch2 were identified.
  • the mitochondria-enriched heavy membrane fraction prepared from 293T cells transfected with HA-BID was treated with cross- linker and then lysed.
  • HA-BID and HA-tBID were clearly visible as a ⁇ 22 kDa and -15 kDa bands (confirmed by mass spectrometry). Mass spectrometry analysis of the -50 kDa band revealed that it included seven peptides from BID ( Figure 11, highlighted in yellow).
  • BID peptides Five BID peptides were not identified and three of them are peptides from tBID ( Figure 11, see peptides that were not highlighted; tBID is generated from cleavage of BID at Asp59 marked in red). A possible reason that these peptides were not identified by the MS analysis is that they were "trapped" by the cross-linker (potential sites of cross- linker binding in one of these peptides are marked in gray).
  • Tables 6 and 7 present peptides from tBID that interact with MTCH2/MIMP.
  • Table 6 Peptides from human BID (SEQ ID NO:4; GenBank Accession No. NPJ)Ol 187) encoded by human BID mRNA (GenBank Accession No. NMJ)Ol 196.2; gi: 37574724; SEQ ID NO:35).
  • Table 7 Peptides from mouse BID (SEQ ID NO:3; GenBank Accession No. AAC71064) encoded by mouse BID mRNA (GenBank Accession No. MMU75506; gi: 1669513; SEQ ID NO:33).
  • the MTCH2/MIMP 240-290 peptide exhibits high affinity towards tBID -
  • the present inventors synthesized peptides and determined their binding affinity toward tBID using fluorescence anisotropy. The results show 2 peptides that bind tBID in the ⁇ M range: MTCH2/MIMP 240-290 (SEQ ID NO: 106;
  • VSNLMAVNNCGLAGGSPPYSPIYTSWIDCWCMLQKAGNMSRGNSLFFRKVP MTCH2/MIMP 140-161
  • SEQ ID NO: 107 Pro Phe His VaI Ile Thr Leu Arg Ser Met VaI GIn Phe He GIy Arg GIu Ser Lys Tyr Cys GIy.
  • the MTCH2/MIMP 240-290 peptide which can be considered as a domain of the C terminal part of Mtch2, exhibits a higher binding affinity to tBID protein relative to the Mtch2 140-161 peptide.
  • the MTCH2/MIMP 240-290 and MTCH2/MIMP 140-161 peptides were labeled with fluorescein at their N terminal, which allows examination of their ability to penetrate cells using a confocal microscope. Both peptides did not penetrate cells, and therefore were further manipulated using a known cell penetrating peptide (penetratinTM peptide; RQIKTWFQNRRMKWKK; SEQ ID NO: 131).
  • Biotinylated MTCH2/MIMP peptides were used to identify BID derived peptides which specifically interact therewith.
  • the bound peptides were identified using avidin conjugated to HRP.
  • the different concentrations of NaCl provide a range of ionic strengths (IS).
  • the array contains peptides derived from six different proteins, of which the first raw are BID-derived peptides. Each peptide array is a double array, with two replicas of the array on the same slide.
  • FIGS. 12A-C show the selective binding of the MTCH2/MIMP 240-290 peptide at a concentration of 20 ⁇ M on the human BID-derived peptide array under 3 different ionic strengths obtained in the presence of 150 mM NaCl (Figure 12A), 100 mM NaCl ( Figure 12B) and 50 mM NaCl ( Figure 12C).
  • the binding results are summarized in Table 8, below.
  • Table 8 Bid-derived peptides that binds MTCH21 MIMP 240-290
  • Table 8 The position of peptide on the array refers to the BID-derived peptide array shown in Figures 12A-C.
  • Figures 13A and 13B demonstrate the binding of MTCH2/MIMP 240-290 peptide in relation to the secondary structure ( Figure 13A) and three-dimensional structure ( Figure 13B) of BID.
  • Figures 14A-C show the selective binding of the MTCH2/MIMP 140-161 peptide at a concentration of 40 ⁇ M on the human BID-derived peptide array under 3 different ionic strengths obtained in the presence of 150 mM NaCl (Figure 14A), 100 mM NaCl ( Figure 14B) and 50 mM NaCl ( Figure 14C).
  • Table 9 The binding results are summarized in Table 9, below.
  • Trp amino acid (W) was added to the peptides in order to measure the peptides concentration using UV spectroscopy for all assays.
  • Figures 15A and 15B demonstrate the binding of MTCH2/MIMP 140-161 peptide in relation to the secondary structure (Figure 15A) and three-dimensional structure (Figure 15B) of BID.
  • MTCH2/MIMP 240-290 domain binds to the following BID peptides:
  • Mtch2 140-161 peptide binds an additional peptide that was not found to bind for MTCH2/MIMP 240-290: 7.
  • tBid 62-76 AA SEQ ID NO: 114;
  • MTCH2/MIMP 140-161 also binds some of the peptides that MTCH2/MIMP 240-290 bound: 1. Full length BID 27-34 AA (SEQ ID NO:110);
  • tBID Full-length human BID is cleaved between D60/G61 (mouse BID is cleaved between D59/G60) to generate tBID, and tBID (61- 195 aa) is the active part that interacts with MTCH2/MIMP and induces cytochrome c release.
  • GNRSSHSRLGRIE SEQ ID NO: 116
  • NRSSHSRLGRIE SEQ ID NO: 115

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Abstract

L'invention porte sur des peptides isolés qui consistent en la séquence d'acides aminés choisie dans le groupe constitué par SEQ ID Nos : 111, 30, 31, 32, 115, 114, 112, 113, 108, 109, 110, 116 et 117, et qui peuvent être utilisés pour augmenter le niveau d'apoptose dans une cellule et pour traiter des pathologies telles que le cancer. L'invention porte également sur des peptides isolés qui consistent en la séquence d'acides aminés présentée dans SEQ ID No :106, 107, 16, 19, 23, 17, 18, 20, 21, 22, 24, 25, 26, 136, 137 ou 138 et qui peuvent diminuer le niveau d'apoptose dans une cellule et traiter des pathologies telles que des maladies neurodégénératives.
PCT/IL2010/000295 2009-04-08 2010-04-08 Peptides isolés pour réguler l'apoptose Ceased WO2010116375A1 (fr)

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WO2014026277A1 (fr) * 2012-08-15 2014-02-20 Université de Montréal Procédé pour l'identification de nouveaux antigènes d'histocompatibilité mineure
WO2016079736A2 (fr) 2014-11-17 2016-05-26 Yeda Research And Development Co. Ltd. Procédés de traitement de maladies associées à une fonction mitochondriale
US10414813B2 (en) 2015-02-09 2019-09-17 Université de Montréal Minor histocompatibility antigens and uses thereof

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