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WO2004038373A2 - Regulation de reponse apoptotique par interaction de proteine a motif tripartite 32 et d'inhibiteurs pias - Google Patents

Regulation de reponse apoptotique par interaction de proteine a motif tripartite 32 et d'inhibiteurs pias Download PDF

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Publication number
WO2004038373A2
WO2004038373A2 PCT/US2003/033599 US0333599W WO2004038373A2 WO 2004038373 A2 WO2004038373 A2 WO 2004038373A2 US 0333599 W US0333599 W US 0333599W WO 2004038373 A2 WO2004038373 A2 WO 2004038373A2
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protein
tripartite motif
pias
motif protein
apoptosis
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WO2004038373A3 (fr
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Amador Albor
Molly Kulesz-Martin
Elizabeth J. Horn
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Oregon Health and Science University
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Oregon Health and Science University
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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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • This disclosure relates to methods for modulating apoptosis, for example in treating a subject having a disorder associated with defects in apoptosis such as cancer or muscular dystrophy. Methods of screening for compounds that can modulate apoptosis are also disclosed.
  • BACKGROUND Cancer is a multistep process comprised of distinct stages, including initiation, promotion, malignant conversion, and progression (Kulesz-Martin, Molecular Mechanisms of chemical and radiation carcinogenesis. Bowden GT and Fischer SM (ed). Pergamon Press: Cambridge, pp. 7-30, 1997). Although these stages likely occur in all human cancers, the individual steps are often unrecognized due to a lack of stage-specific markers. Evidence from retmoblastoma (Knudson, Proc. Natl Acad. Sci.
  • TR 20 PYRIN/MARENOSTRIN
  • TRHVI18 Modline one/MLDl
  • TRLM37 Multiple/MUL
  • familial Mediterranean fever X-linked Opitz/GBBB syndrome
  • mulibrey nanism dwarfism
  • TRBVI19 Promyelocytic leukemia/PML
  • TRIM27 Ret finger protein/RFP
  • TRIM24 Transcriptional intermediary factor 1/TLF1
  • RAR ⁇ Retinoic acid receptor alpha
  • RET Ret proto-oncogene
  • B-RAF v-raf murine sarcoma viral oncogene homologBl
  • TRDVI25 and Trim32 are unique among TRIM proteins in being linked to cancer without being oncogenic fusion proteins. Further, the
  • TRIM32 gene is mutated in Limb-Girdle Muscular Dystrophy type 2H (LGMD2H), a mild autosomal recessive myopathy (Frosk et al, Am. J.Hum. Genet. 70:663-72, 2002).
  • TRIM32 contains several conserved domains.
  • the RING and B-Box domains are zinc fingers with conserved cysteine residues.
  • RING domains are present in E3-ubiquitin ligases and mediate interaction with E2-ubiquitin conjugating enzymes (Joazeiro and eissman, Cell 102:549-52, 2000), while the coiled-coil domain mediates homo- and heterodimerization (Lupas, Trends Biochem.Sci. 21:375-82, 1996).
  • TREV132 also contains a carboxy-terminal NHL domain (Slack and Ruvkun, Trends Biochem.Sci. 23:474-5, 1998).
  • RLNG domains are characteristic of proteins with E3-ubiquitin ligase activity, including proteins involved in the control of apoptosis (cIAPl, cIAP2, XIAP and all TRAF proteins except TRAF1), transcription (Sina, Rbxl and Mdm2), cell cycle (APC11), tyrosine kinase growth factor receptor signaling (CBL family members), and the tumor suppressor protein BRCA1 (Joazeiro and Weissman, Cell 102:549-52, 2000).
  • TRIM proteins are E3-ubiquitin ligases, including TRLM18, which targets the degradation of phosphatase 2A, PP2Ac (Trockenbacher et al, Nat. Genet. 29:287-94, 2001), and TRLM25, which targets the degradation of 14-3- 3- ⁇ (Urano et al, Nature 417:871-5, 2002).
  • TRLM18 targets the degradation of phosphatase 2A, PP2A
  • tripartite motif protein 32 such as human TRLM32 and mouse Trim32
  • HNSCC human head and neck squamous cell carcinomas
  • tripartite motif protein 32 interacts and ubiquitylates protein inhibitors of activated signal transducer and activator of transcription (PIAS) to promote PIAS degradation.
  • PIAS activated signal transducer and activator of transcription
  • LGMD2H limb-girdle muscle dystrophy type 2H
  • Methods of increasing apoptosis can be used to treat disorders associated with decreased apoptosis, for example atopic dermatitis, and hyperproliferative disorders such as tumors and cancers, such as skin cancer and leukemia.
  • Methods of decreasing apoptosis can be used to treat disorders associated with increased apoptosis, for example muscular dystrophy, ischemia or the CNS side effects of interferon therapy.
  • specific binding agents that specifically recognize a tripartite motif protein 32 protein, and interfere with its cellular activity.
  • specific binding agents include, but are not limited to antibodies, such as polyclonal antibodies, monoclonal antibodies, and humanized antibodies.
  • FIG. 1 is a schematic diagram of the cellular relationships within the mouse clonal model of epidermal carcinogenesis.
  • FIG. 2 is a schematic drawing showing an amino acid sequence alignment of human TRIM 32 (SEQ LD NO: 3) and mouse Trim32 (SEQ LD NO: 2) proteins.
  • SEQ LD NO: 3 human TRLM32 protein sequence is shown, with differences in a mouse sequence indicated below.
  • Homozygous mutation of the aspartic acid residue 487 to asparagine (in mouse, D489N) found in LGMD2H is marked with a box.
  • FIG. 3 is a bar graph showing Trim32 mRNA relative expression levels relative to normal human liver. Trim32 relative expression of normal mucosa samples is shown in the inset. Each sample was run in triplicate per plate (3 plates total), and error bars depict standard deviation across mean the value of three plates. Patients indicated with an asterisk had statistically significant Trim32 expression levels between tumor and uninvolved mucosa tissue (p ⁇ 0.05) using a two-tailed student's T-test.
  • FIG. 4A is a schematic drawing showing a summary of an in vitro transformation assay used to measure Trim32 activity in carcinogenesis.
  • FIG. 4B is a bar graph showing the results of an in vitro transformation assay demonstrating that Trim32 increases transformation frequency. Transformation frequencies (%TF) were normalized to GFP %TF. Error bars represent standard error of the mean in the 3 replicate assays. Differences in %TF between Trim32 and GFP were statistically significant, p ⁇ .0001 Wilcoxon rank sum test.
  • FIG. 5 is a graph showing Kaplan Meier analysis of annular plaque formation in p53 -/- Trim32 and p53 -/- GFP groups in days.
  • FIG. 6A is a bar graph showing the percent of cells undergoing apoptosis in the presence or absence of Trim32, demonstrating that Trim32-expressing cells are less sensitive to TNF ⁇ /UNB treatment. Statistically significant differences (indicated) were tested by single tailed Wilcoxon Rank Sum test.
  • FIG. 6B is a bar graph showing the percent of sunburn cells in GFP grafts following exposure to UNB (doses of 600 and 1200 J/m 2 ), in the presence or absence of Trim32. Statistical differences (indicated) were calculated using the Wilcoxon Rank Sum Test.
  • FIG. 7A is a schematic drawing showing the relative amount of binding between various Trim32 constructs and Pias ⁇ .
  • FIG. 7B is a schematic drawing of human PIASy and mouse Pias ⁇ genes.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. If only one strand of a nucleic acid sequence is shown, the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 shows a mouse Trim32 nucleic acid coding sequence.
  • SEQ LD NO: 2 shows a mouse Trim32 amino acid sequence encoded by SEQ ID NO: 1.
  • SEQ ID NO: 3 shows a human TRLM32 amino acid sequence.
  • SEQ K ) NOS: 4 and 5 show nucleotide primers specific for human TRTM32 used in quantitative real-time PCR (qPCR) assays.
  • SEQ ID NOS: 6 and 7 show nucleotide primers specific for 18S used in quantitative real-time PCR (qPCR) assays.
  • SEQ ID NO: 8 shows a human PIASy cDNA sequence.
  • SEQ ID NO: 9 shows a mouse Pias ⁇ cDNA sequence.
  • SEQ ID NO: 10 shows a human PIASy amino acid sequence.
  • SEQ ID NO: 11 shows a mouse Pias ⁇ amino acid sequence.
  • SEQ ID NO: 12 shows a human TR 32 nucleic acid sequence
  • dsRNA double-stranded RNA
  • GFP green fluorescent protein
  • HNSCC head and neck squamous cell carcinoma
  • LGMD2H Limb-Girdle Muscular Dystrophy type 2H
  • SBC sunburn cell
  • SCC squamous cell carcinoma
  • ssRNA single-stranded RNA
  • TNF ⁇ tumor necrosis factor alpha
  • TPA 12- O-tetradecanoylphorbol-13-acetate
  • UVB ultraviolet B Agent: Any substance, including, but not limited to, a chemical compound, antibody, chemotherapeutic, hormone, nucleotide sequence, peptide mimetic, protein, sugar, lipid, and the like.
  • Immunoglobulin (Ig) molecules and immunologically active portions of Ig molecules such as molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
  • Monoclonal and polyclonal immunoglobulin, as well as immunologically effective portions ("fragments") thereof, are encompassed by the disclosure.
  • Antibodies disclosed herein can also be humanized mAbs (or immunologically effective portions thereof).
  • An exemplary immunoglobulin is IgG.
  • Naturally occurring IgG includes four polypeptide chains, two heavy chains and two light chains inter-connected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody.
  • antigen-binding fragments are also intended to be designated by the term "antibody".
  • binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the variable light (NL), variable heavy (NH), constant light (CL) and constant heavy (CH)1 domains; (ii) an Fd fragment consisting of the NH and CHI domains; (iii) an Fv fragment consisting of the NL and NH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al, Nature 341:544-6, 1989) which consists of a NH domain; and (v) an F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al, Science 242:423- 6, 1998 and Huston efal, Proc. Natl Acad. Set 85:5879-83, 1988) by recombinant methods.
  • single chain Fv single chain Fv
  • dsFv disulfide stabilized Fv
  • dimeric Fvs diabodies
  • An antibody further includes humanized and chimeric molecules that specifically bind the target antigen.
  • PIASy antibodies are known in the art (for example hngenex Corp. Cat. No. IMG-290).
  • antibodies can also be produced using standard procedures, for example as described in Harlow and Lane (Antibodies: A Laboratory Manual. 1988).
  • "Specifically binds" refers to the ability of individual antibodies to specifically immunoreact with an antigen. This binding is a non-random binding reaction between an antibody molecule and the antigen.
  • Binding specificity is typically determined from the reference point of the ability of the antibody to differentially bind the antigen of interest and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes.
  • An antibody that specifically binds to a particular epitope is referred to as a "specific antibody”.
  • detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as P, fluorophores, chemiluminescent agents, and enzymes.
  • Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA.
  • Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA.
  • Antigene molecules are either antisense or sense molecules directed to a dsDNA target.
  • Double-stranded DNA (“dsDNA”) has two strands, a 5'-» 3' strand, referred to as the plus strand, and a 3' -» 5' strand (the reverse complement), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' -> 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T).
  • Apoptosis A form of cell death induced by external stimuli that activates a proteolytic activation cascade of intracellular proteases called caspases.
  • the resulting signaling events lead (through the activation of caspases) to the digestion of numerous cellular protein substrates, such as permeabilization of the mitochondia with release of intramitochondrial components, including cytochrome C, and the fragmentation of genomic DNA into small molecules about hundreds of nucleotides long.
  • These changes are incompatible with cellular viability, and create distinct microscopic features, for example, chromatin condensation and fragmentation. Cells exhibiting these microscopic features are called apoptotic bodies.
  • Apoptosis can be quantified by a variety of methods, both microscopic (apoptotic body count, see Example 6) and biochemical (determination of caspase activation).
  • Binding or Interaction Formation of a complex between a protein or nucleic acid with one or more other proteins, nucleic acids or other agents such as therapeutic compounds. Generally, the stronger the binding of the molecules in the complex, the slower their rate of dissociation. The strength of the bonding between two molecules can be used to index the specificity of the recognition between the molecules.
  • a ⁇ protein sequence such as a peptide, binds or stably binds to another protein or other agent if a sufficient amount of the protein forms chemical bonds to another protein or other agent to permit detection of that binding.
  • Non-limiting methods of detecting such complex formation include immunoprecipitation followed by immunoblotting (for example using the methods disclosed in Examples 8 and 9), and yeast and mammalian two hybrid systems (see Example 8).
  • a nucleotide sequence such as a nucleotide probe or primer, binds or stably binds to a target nucleic acid if a sufficient amount of the nucleotide sequence forms base pairs or is hybridized to its target nucleic acid to permit detection of that binding. Binding can be detected by either physical or functional properties of the targe nucleotide sequence complex. Binding between a target and a nucleotide sequence can be detected by any procedure, including but not limited to functional and physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like.
  • Physical methods of detecting the binding of complementary strands of DNA or RNA include such methods as DNAse I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method involves observing a change in light absorption of a solution containing a nucleotide sequence (or an analog) and a target nucleic acid at 220 to 300 ⁇ m as the temperature is slowly increased. If the nucleotide sequence or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt.
  • cDNA complementary DNA: A piece of DNA lacking internal, non- coding segments (introns) and regulatory sequences that determine transcription. cDNA can be synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • a therapy decreases apoptosis if detectable apoptosis is reduced as compared to apoptosis in the absence of the therapy.
  • increased levels of tripartite motif protein 32 decrease apoptosis, for example decreases apoptosis of a cell in a subject in whom decreased apoptosis is desired, for example a subject having muscular dystrophy.
  • Such reduction can be measured, for example, by microscopy as described in EXAMPLE 6.
  • a therapy decreases an interaction between tripartite motif protein 32 and PIAS, thereby increasing apoptosis, such as a cell in a subject having a disorder associated with decreased apoptosis, for example a subject having cancer or atopic dermatitis, if the relative amount of apoptosis is increased as compared to the amount of apoptosis in the absence of the therapy.
  • DNA deoxyribonucleic acid: A long chain polymer which includes the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)).
  • the repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule.
  • a reference to the nucleic acid molecule that encodes a tripartite motif protein 32, or a fragment thereof, encompasses both the sense strand and its reverse complement.
  • a nucleotide sequence is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • Enhance or increase To improve the quality, amount, or strength of something.
  • a therapy enhances or increases apoptosis, if the relative amount of apoptosis increases in the presence of a therapy, such as a therapy that decreases an interaction between tripartite motif protein 32 and PIAS.
  • agents that decrease tripartite motif protein 32 expression such as expression of human TRLM32 or mouse Trim32
  • increase PIAS activity or decrease an interaction between tripartite motif protein 32 and PIAS enhance apoptosis, for example increase apoptosis in a subject, such as a subject having photodamage or radiation damage, skin cancer, or other cancer such as leukemia, or atopic dermatitis.
  • Such enhancement can be measured using any bioassay known in the art, for example, using microscopy as described in EXAMPLE 6.
  • a therapy enhances an interaction between a tripartite motif protein 32 and PIAS, if such interaction increases as compared to the relative amount of interaction in the absence of the therapy, hi a particular example, agents that increase tripartite motif protein 32 expression (such as expression of human TRIM32 or mouse Trim32) or increase tripartite motif protein 32 interaction with PIAS (such as human PIASy or mouse Pias ⁇ ) decrease apoptosis, for example in a subject in whom decreased apoptosis is desired, for example in a subject having muscular dystrophy, ischemia or the CNS side effects of interferon therapy.
  • Such enhancement of interaction can be measured, for example, using the binding assays described in EXAMPLE 8.
  • Functional deletion or disruption A deletion or mutation of a nucleic acid or amino acid sequence that substantially decreases the biological activity of the nucleic acid or amino acid sequence.
  • the function of a gene or gene product is reduced or eliminated by a deletion, insertion, or substitution.
  • functional deletion of tripartite motif protein 32 such as human TRIM32 or mouse Trim32, reduces or can even eliminate detectable tripartite motif protein 32 activity, such as the ability of tripartite motif protein 32 to reduce apoptosis.
  • tripartite motif protein 32 functional deletion is the D489N (mouse) or D487N (human) tripartite motif protein 32 mutation found in LGMD2H patients (see FIG. 2).
  • Interfering with or inhibiting (expression of a target gene) The ability of an agent, such as an interfering nucleotide sequence, such as a dsRNA, siRNA, antisense, or other molecules, to measurably reduce the expression of a target gene. It contemplates reduction of the end-product of the gene, such as the expression or function of the encoded protein, and thus includes reduction in the amount or longevity of the mRNA transcript.
  • an agent such as an interfering nucleotide sequence, such as a dsRNA, siRNA, antisense, or other molecules
  • the gene is expressed at least 5% less than prior to application of the agent, for example at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 50% less, at least 75% less, or even at least 95% less, or even more.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.
  • Modulate To increase or decrease. For example, a therapy that results in modulation of apoptosis of a cell increases or decreases apoptosis of the cell or a population of cells, as compared to an amount of apoptosis in the absence of the therapy.
  • Nucleic acid A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and encompasses analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • a nucleotide is one monomer in a nucleotide sequence or an oligonucleotide/polynucleotide.
  • Oligonucleotide A linear polynucleotide (such as DNA or RNA) sequence of at least 9 nucleotides, for example at least 15, 18, 24, 25, 27, 30, 50, 100 or even 200 nucleotides long.
  • ORF open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Protein inhibitors of activated signal transducer and activator of transcription PIAS
  • the term PIAS includes any PIAS protein from any organism that has the property of stimulating or increasing apoptosis, and can bind to tripartite motif protein 32.
  • PIAS nucleic acids for example PIAS genes, cDNAs and RNAs that encode PIAS.
  • PIAS molecules can sumoylate Smad3 and Smad4.
  • PIAS is downregulated in later stages of chronic myelogenous leukemia, and expression of PIAS in these stages induces apoptosis.
  • Examples of native PIAS nucleic acid sequences include, but are not limited to: Genbank Accession Nos: NM__004364 and BC010047 (human PIASy, SEQ LD NO: 8) and NM_021501 and BC025159 (mouse Piasy; SEQ ID NO: 9).
  • Examples of PIAS amino acid sequences include, but are not limited to: Genbank Accession Nos: AAH29874 and NP_001946 (human PIASy; SEQ ID NO: 10) and Q9JM05 and NP_067476 (mouse Piasy; SEQ ID NO: 11).
  • a PIAS sequence includes a full-length wild-type (or native) sequence, as well as PIAS allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to increase apoptosis.
  • PIAS has at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to a native PIAS, and retains the property of stimulating or increasing apoptosis.
  • PIAS activity The activity of PIAS agents in which the amount of cellular apoptosis is affected.
  • PIAS agents include, but are not limited to PIAS proteins, such as human PIASy or mouse Pias ⁇ (including variants, fusions, fragments and mimetics thereof), nucleic acids (including DNA, RNA, siRNA, and antisense molecules), specific binding agents, agonists, and antagonists.
  • PIAS activity occurs when PIAS proteins, nucleic acids, specific binding agents, and/or agonists increase apoptosis of a cell or group of cells, for example by at least 10%, for example by at least 25%, at least 50%, or even at least 75%, as compared to an amount of apoptosis in the absence of the agent.
  • PIAS activity is reduced or decreased when PIAS proteins, nucleic acids, specific binding agents, and/or antagonists decrease apoptosis of a cell or group of cells, for example by at least 10%, for example by at least 25%, at least 50%, or even at least 75%, as compared to an amount of apoptosis in the absence of the agent.
  • PIAS protein such as human PIASy or mouse Pias ⁇
  • a PIAS protein can be assessed for its ability to increase apoptosis by contacting a cell with the protein, for example administering the protein to a subject in need of increased apoptosis.
  • Functional protein activity would be detected by an increase in apoptosis of undesired cells, such as cancer cells, in the presence of the protein.
  • Any of these assays can be modified by using in vivo expression a PIAS gene, and variants, fusions, and fragments thereof, instead of administering purified proteins.
  • Polynucleotide A linear nucleic acid sequence of at least 3 nucleotides. Therefore, a polynucleotide includes molecules which are at least 15, 20, 30, 50, 100, 200, 500, 1000, or 5000 nucleotides in length, and also nucleotides as long as a full length cDNA. For example, a TRTM32 polynucleotide encodes a TRIM32 peptide, while a PIASy polynucleotide encodes a PIASy peptide.
  • Polypeptide Any chain of amino acids at least six amino acids in length, such as at least 8 amino acids, such as at least 9 amino acids, such as about 10-100 amino acids, regardless of post-translational modification (such as glycosylation or phosphorylation).
  • Preventing disease A therapeutic intervention that inhibits the full development of a disease, for example preventing development of cancer in a subject having UNB damage to the skin, or preventing the C ⁇ S side effects of interferon therapy.
  • a probe includes an isolated nucleic acid sequence attached to a detectable label or other reporter molecule.
  • Typical labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
  • Primers are short nucleic acid molecules, for instance DNA oligonucleotides 9 nucleotides or more in length, for example that hybridize to contiguous complementary nucleotides or a sequence to be amplified. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme.
  • Primer pairs can be used for amplification of a nucleic acid sequence, for example by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription- free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in 5,427,930; andNASBATM RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134.
  • Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure. It is also appropriate to generate probes and primers based on fragments or portions of these disclosed nucleic acid molecules, for instance regions that encompass mutations in a tripartite motif protein 32 coding sequence, for example a TRIM32 or Trim32 coding sequence.
  • Amplification primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Nersion 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • a primer including 20 consecutive nucleotides of a TRIM32-encoding nucleotide sequence or flanking region thereof will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides.
  • Promoter An array of nucleic acid control sequences that directs transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (Bitter et al, Meth. Enzymol 153:516-44, 1987).
  • promoters include promoters derived from the genome of mammalian cells (such as a metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used to practice the disclosed methods. Promoters produced by recombinant D ⁇ A or synthetic techniques can also be used. For example, a polynucleotide encoding a therapeutic protein, such as TRLM32 or PIASy, can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its environment within a cell, such that the peptide is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that may accompany it.
  • a purified peptide preparation is one in which the peptide is substantially-free from contaminants, such as those that might be present following chemical synthesis of the peptide.
  • a TRLM32 peptide is purified when at least 60% by weight of a sample is composed of the peptide, for example when 75%, 95%, or 99% or more of a sample is composed of the peptide.
  • methods that can be used to purify an antigen include, but are not limited to the methods disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 17). Protein purity can be determined by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high-pressure liquid chromatography; sequencing; or other conventional methods.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example by genetic engineering techniques.
  • a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
  • RNA ribonucleic acid
  • messenger which encodes proteins
  • rRNA ribosomal
  • tRNA transfer molecules responsible for transferring amino acid monomers to the ribosome during protein synthesis
  • Messenger RNA includes heteronuclear (hnRNA) and membrane- associated polysomal RNA (attached to the rough endoplasmic reticulum).
  • Total RNA refers to a heterogeneous mixture of all types of RNA molecules.
  • Sequence identity/similarity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (for example, human and mouse sequences), compared to species more distantly related (for example human and C. elegans sequences).
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11 , and a per residue gap cost of 1).
  • tripartite motif protein 32 or PIAS homologs are typically characterized by possession of at least 70% sequence identity counted over the full- length alignment with a tripartite motif protein 32 or PIAS amino acid sequence, respectively, using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).
  • Tripartite motif protein 32 or PIAS proteins with even greater similarity to the reference sequence show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, at least 90%, at least 95% or at least 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
  • nucleic acid molecules that hybridize under stringent conditions to a tripartite motif protein 32 gene sequence typically hybridize to a probe based on either an entire tripartite motif protein 32 gene or selected portions of the gene.
  • An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • Nucleic acid sequences that do not show a high degree of identity to a tripartite motif protein 32 or PIAS sequence may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a tripartite motif protein 32 or PIAS nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein, and can be used in the disclosed methods. Such homologous nucleic acid sequences can, for example, possess at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity determined by this method.
  • Short/Small Interfering Nucleotide Sequence A nucleotide sequence capable of interfering with gene expression, for instance by inducing gene- specific inhibition of expression.
  • sequence of a siRNA is substantially identical to a portion of a transcript of a target gene (mRNA) for which interference or inhibition of expression is desired.
  • small, double stranded RNAs of about 15 to about 40 nucleotides in length (the length of each of the individual strands of the dsRNA), such as about 20 to about 25 nucleotides in length, and in some examples about 23 nucleotides in length, that interfere with, or inhibit, expression of a target sequence.
  • the RNA backbone and/or component nucleotides can be unmodified or modified.
  • the dsRNA can contain one or more deoxy-nucleic acids.
  • Synthetic small dsRNAs may be used to induce gene-specific inhibition of expression.
  • the dsRNAs can be formed from complementary single stranded RNAs ("ssRNAs") or from a ssRNA that forms a hairpin or from expression from a DNA vector.
  • ssRNAs complementary single stranded RNAs
  • these small interfering nucleotide sequences have 3' and/or 5' overhangs on each strand of the duplex. These overhangs can be 0 nucleotides (that is, blunt ends) to 5 nucleotides in length.
  • siRNA molecules can be used as reverse genetic and therapeutic tools in mammalian cells, including human cells, both in vitro and in vivo.
  • These small interfering nucleotide sequences are suitable for interference or inhibition of expression of a target gene wherein the sequence of the small interfering nucleotide sequence is substantially identical to a portion of an mRNA or transcript of the target gene for which interference or inhibition of expression is desired.
  • nucleotides suitable for inhibiting or interfering with the expression of a target sequence include nucleotide derivatives and analogs.
  • a non-natural linkage between nucleotide residues can be used, such as a phosphorothioate linkage.
  • the nucleotide strand can be derivatized with a reactive functional group or a reporter group, such as a fluorophore.
  • the 2'-hydroxyl at the 3' terminus can be readily and selectively derivatized with a variety of groups.
  • nucleotide derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2'-O-alkylated residues or 2'-deoxy- 2'-halogenated derivatives.
  • modified carbohydrate moieties include 2'-O-methyl ribosyl derivatives and 2'-O-fluoro ribosyl derivatives.
  • the nucleotide bases can be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated.
  • the bases may also be alkylated, for example, 7-methylguanosine may be incorporated in place of a guanosine residue.
  • Non-natural bases that yield successful inhibition can also be incorporated.
  • a tripartite motif protein 32-specific binding agent binds substantially only to a tripartite motif protein 32 protein.
  • a particular tripartite motif protein 32-specific binding agent is specific for one form of tripartite motif protein 32, such as a mutated form of tripartite motif protein 32, and are capable of distinguishing mutated tripartite motif protein 32 from the wild- type.
  • One such mutated form of tripartite motif protein 32 is the LGMD2H mutated form of tripartite motif protein 32 (D487N in human TRBVI32 and D489N in mouse
  • a tripartite motif protein 32 specific binding agent interferes with binding between tripartite motif protein 32 and PIAS.
  • apoptosis of the cell increases.
  • such an agent is administered to a subject in need of increased apoptosis.
  • specific binding agent includes shorter fragments of antibodies that can also serve as specific binding agents. For instance, Fabs, Fvs, and single- chain Fvs ("scFvs"). Methods of making these fragments are routine.
  • Subject Living multicellular vertebrate organisms, a category which includes both human and veterinary subjects for example, mammals, rodents, and birds.
  • Therapeutically active molecule An agent, such as a tripartite motif protein 32 or PIAS protein, nucleic acid, agonist or antagonist, that can modulate apoptosis as measured by clinical response (for example a decrease in the number of cancer cells or tumor size, or an increase in the number myoblasts). In some examples, it is an agent that increases or decreases an interaction between a tripartite motif protein 32 and PIAS as measured by clinical response (for example increasing or decreasing the number of cells). In other examples, it is an agent that decreases cellular activity of tripartite motif protein 32 to increase apoptosis. hi yet other examples, it is an agent that increases cellular activity of tripartite motif protein 32 to decrease apoptosis.
  • a tripartite motif protein 32 or PIAS protein nucleic acid, agonist or antagonist
  • Therapeutically active molecules can be made from nucleic acids.
  • nucleic acid based therapeutically active molecules are a nucleic acid sequence that encodes a tripartite motif protein 32 protein, or PIAS wherein the nucleic acid sequence is operably linked to a control element such as a promoter.
  • Other non- limiting examples of nucleic acid therapeutic molecules are tripartite motif protein 32 or PIAS antisense molecules, ribozymes, or siRNAs.
  • Other therapeutically active molecules include proteins as well as organic or other chemical compounds that mimic the effects of a peptide, such as tripartite motif protein 32 or PIAS, which increase or decrease an interaction between tripartite motif protein 32 and PIAS, or which can increase or decrease the cellular activity of tripartite motif protein 32 to modulate apoptosis.
  • a peptide such as tripartite motif protein 32 or PIAS, which increase or decrease an interaction between tripartite motif protein 32 and PIAS, or which can increase or decrease the cellular activity of tripartite motif protein 32 to modulate apoptosis.
  • Therapeutically effective amount A quantity of an agent sufficient to achieve a desired effect in a subject or a cell being treated. For instance, this can be the amount necessary to inhibit or to measurably reduce tripartite motif protein 32 activity in a cell (such as TRTM32 or Trim32 activity), increase PIAS activity in a cell (such as PIASy or Pias ⁇ activity), or decrease an interaction between tripartite motif protein 32 and PIAS, for example to increase apoptosis.
  • tripartite motif protein 32 activity in a cell such as TRTM32 or Trim32 activity
  • decrease PIAS activity in a cell such as PIASy or Pias ⁇ activity
  • increase an interaction between tripartite motif protein 32 and PIAS such as TRLM32/PIASy or Trim32/Pias ⁇
  • a desired response can be a decrease in apoptosis.
  • a therapeutic effect is an increase in the number of cells, such as myoblast cells, in a subject having LGMD2H.
  • a desired response can be an increase in apoptosis.
  • a therapeutic effect is a decrease in the number of cells, such as cancers cells, which can lead to a regression of a cancer, such as skin cancer or leukemia.
  • a therapeutically effective amount of an agent may be administered in a single dose, or in several doses, for example daily or more often, during a course of treatment. However, the effective amount will be dependent on the particular agent applied, the subject being treated, the severity and type of the affliction, and the manner of administration.
  • a therapeutically effective amount of a tripartite motif protein 32 or PIAS protein can vary from about 0.01 mg/kg body weight to about 1 g/kg body weight, such as about 1 mg per subject.
  • the therapeutic agents disclosed herein can be administered with other biologically active agents, for example chemotherapeutic agents, corticosteroids, and antihistamines.
  • a virus or vector "transduces” or “transfects” a cell when it transfers nucleic acid into the cell.
  • a cell is
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transgene An exogenous nucleic acid sequence supplied by a vector.
  • a transgene encodes a tripartite motif protein 32 or PIAS polypeptide.
  • Treating a disease A therapeutic intervention that ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop. Includes inhibiting or preventing the partial or full development or progression of a disease or medical condition or abnormal biological state in a subject, for example in a person who is known to have a predisposition to or to be at risk for the disease or medical condition or abnormal biological state. Examples of such conditions include, but are not limited to atopic dermatitis, a cancer such as skin cancer or leukemia, muscular dystrophy, ischemia, or the CNS side effects of interferon therapy.
  • the therapeutic intervention can be prophylactic inhibition of a disease or medical condition or biological state, and therapeutic interventions to alter the natural course of an untreated disease process or medical condition or a biological state, such as a tumor growth.
  • the therapeutic intervention can be administration of a biological agent that modulates apoptosis, systemically, regionally, or topically or locally.
  • Tripartite motif protein 32 includes any tripartite motif protein 32 gene, cDNA, RNA, or protein from any organism that has the property of decreasing apoptosis. In some examples, tripartite motif protein 32 increases tumor activity. Typically, full-length tripartite motif protein 32 proteins include the following domains: a RTNG domain, a B-box, a coiled-coil domain characteristic of the tripartite motif (TRIM) family, and a carboxy-terminal NHL domain. Exemplary tripartite motif protein 32 molecules include human TRLM32 sequences and mouse Trim32 sequences, as well as variants, fragments and fusions thereof that retain tripartite motif protein 32 activity.
  • Examples of native tripartite motif protein 32 nucleic acid sequences include, but are not limited to: Genbank Accession Nos: NM_012210 (human; SEQ LD NO: 12) and AF347694 (mouse; SEQ ID NO: 1).
  • Examples of tripartite motif protein 32 amino acid sequences include, but are not limited to: Genbank Accession Nos: NP_036342 (human; SEQ ID NO: 3) and NM_053084 (mouse; SEQ ID NO: 2).
  • a tripartite motif protein 32 sequence includes a full-length wild-type (or native) sequence, as well as tripartite motif protein 32 allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to decrease apoptosis.
  • tripartite motif protein 32 has at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to a native tripartite motif protein 32 sequence, and retains the property of decreasing apoptosis.
  • Tripartite motif protein 32 activity The activity of tripartite motif protein 32 agents in which the amount of cellular apoptosis is affected. Tripartite motif protein 32 agents include, but are not limited to tripartite motif protein 32 proteins (including variants, fusions, fragments and mimetics thereof), nucleic acids
  • RNA including DNA, RNA, siRNA, and antisense molecules
  • specific binding agents including DNA, RNA, siRNA, and antisense molecules
  • agonists including DNA, RNA, siRNA, and antagonists.
  • tripartite motif protein 32 activity occurs when tripartite motif protein 32 proteins, nucleic acids, specific binding agents, or agonists decrease apoptosis, for example by at least 10%, for example by at least 25%, at least 50%, or even at least 75%, as compared to an amount of apoptosis in the absence of the agent.
  • Tripartite motif protein 32 activity is reduced or decreased when tripartite motif protein 32 proteins, nucleic acids, specific binding agents, or antagonists increase apoptosis of a cell or group of cells, for example by at least 10%, for example by at least 25%, at least 50%, or even at least 75%, as compared to an amount of apoptosis in the absence of the agent.
  • a tripartite motif protein 32 protein (such as TRIM32 or Trim32) can be assessed for its ability to decrease apoptosis by contacting a cell with the protein, for example administering the protein to a subject in need of decreased apoptosis, such as a subject having LGMD2H.
  • Functional protein activity would be detected by a decrease in apoptosis of desired cells, such as myoblast cells, in the presence of the protein.
  • Any of these assays can be modified by using in vivo expression a tripartite motif protein 32 gene, and variants, fusions, and fragments thereof, instead of administering purified proteins.
  • tripartite motif protein 32 imparts a survival phenotype to epidermal cells responding to TNF ⁇ /UNB-induced stress, whereby these epidermal cells persist and can accumulate additional UVB- induced D ⁇ A damage or other oncogenic events, leading to cancer development. It is also shown that tripartite motif protein 32 binds and ubiquitylates PIAS to promote PIAS degradation, hi particular examples, it is illustrated that Trim32 binds and ubiquitylates Pias ⁇ , the mouse ortholog to human PIASy, promoting Pias ⁇ degradation.
  • the limb-girdle muscle dystrophy type 2H (LGMD2H) mutation of tripartite motif protein 32 prevents interaction of tripartite motif protein 32 and PIAS.
  • Trim32 proteins having the LGMD2H mutation eliminated detectable binding to Pias ⁇ . fr activation of tripartite motif protein 32 in LGMD2H, such as human TRIM32 or mouse Trim32, allows PIAS (such as PIASy or Pias ⁇ ) to activate death signals, resulting in myopathy. That is, the lack of tripartite motif protein 32 function in LGMD2H increases apoptosis, which could be responsible for the muscle wasting phenotype observed in LGMD2H patients.
  • tripartite motif protein 32 activation promotes carcinogenesis by blocking certain stress-induced apoptotic signaling pathways, while inactivation of tripartite motif protein 32 signaling exacerbates apoptotic signaling in muscle dystrophy.
  • tripartite motif protein 32 mediated modification of PIAS is likely enhanced by cellular stress signals (such as UVB and T ⁇ F ⁇ in keratinocytes), and represents a survival enhancing mechanism, whereby reduction of PIAS levels decreases apoptotic potential.
  • Methods are disclosed for modulating apoptosis in a cell, for example increasing or decreasing apoptosis of cells.
  • the method includes modulating an interaction between a tripartite motif protein 32 and PIAS in the cell, wherein modulating this interaction modulates apoptosis in the cell.
  • a subject is administered an effective amount of an agent that decreases cellular activity of tripartite motif protein 32 to increase apoptosis, or administered an agent that increases cellular activity of tripartite motif protein 32 to decrease apoptosis.
  • the tripartite motif protein 32 can be any tripartite motif protein 32 from any organism, such as a human tripartite motif protein 32 (TRJM32), or a mouse tripartite motif protein 32 (Trim32), as well as variants, fragments, and fusions thereof that retain tripartite motif protein 32 biological activity.
  • exemplary tripartite motif protein 32 proteins include, but are not limited to, sequences including at least 70% sequence identity to SEQ ID NOS: 2 or 3, such as at least 80%, at least 90%, at least 95%, at least 98% or even at least 99% sequence identity to SEQ ID NOS: 2 or 3.
  • a tripartite motif protein 32 protein includes SEQ ID NO: 2 or 3.
  • the PIAS protein can be any PIAS protein from any organism, such as a human PIAS (PIASy), or a mouse PIAS (Pias ⁇ ), as well as variants, fragments, and fusions thereof that retain PIAS biological activity.
  • PIAS proteins include, but are not limited to, sequences including at least 70% sequence identity to SEQ LD NOS: 10 or 11, such as at least 80%, at least 90%, at least 95%, at least 98% or even at least 99% sequence identity to SEQ LD NOS: 10 or 11.
  • a PIAS protein includes SEQ LD NO: 10 or 11.
  • Such proteins can be encoded by a PIAS nucleic acid, such as a nucleic acid including at least 70% sequence identity to SEQ LD NOS: 8 or 9, such as at least 80%, at least 90%, at least 95%, at least 98% or even at least 99% sequence identity to SEQ LD NOS: 8 or 9.
  • PIAS nucleic acid includes SEQ ID NO: 8 or 9.
  • Apoptosis can be modulated by increasing apoptosis, for example by decreasing an interaction between tripartite motif protein 32 and PIAS proteins, or by administering an agent that decreases cellular activity of tripartite motif protein 32.
  • Increasing apoptosis can be used to treat a subject having a disorder characterized by decreased apoptosis, for example cellular hyperproliferation. Examples of such disorders include, but are not limited to atopic dermatitis, a tumor, or cancer, such as skin cancer or leukemia.
  • the interaction between tripartite motif protein 32 and PIAS can be decreased by contacting a cell with an agent that decreases an amount of tripartite motif protein 32 in the cell.
  • agents that decrease the amount of cellular tripartite motif protein 32 include tripartite motif protein 32 antisense molecules, ribozymes, and siRNA molecules.
  • Mutant tripartite motif protein 32 proteins or nucleic acid sequences encoding such mutant tripartite motif protein 32 sequences can also be used to decrease levels of cellular tripartite motif protein 32.
  • mutant tripartite motif protein 32 sequences include, but are not limited to, those that decrease the binding between tripartite motif protein 32 and PIAS, such as SEQ ID NO: 2 having a D489N mutation or SEQ ID NO: 3 having a D487N mutation.
  • Specific-binding agents such as antibodies, can also be used to decrease the interaction between the tripartite motif protein 32 and PIAS protein.
  • Agents that can be used to decrease cellular activity of tripartite motif protein 32 include, but are not limited to, agents that decrease the interaction between tripartite motif protein 32 and PIAS, specific binding agents that bind to tripartite motif protein 32 or PIAS, and nucleic acid sequences that interfere or disrupt tripartite motif protein 32 expression.
  • Apoptosis can be modulated by decreasing or reducing apoptosis, for example by increasing an interaction between tripartite motif protein 32 and PIAS proteins or by administering an agent that increases cellular activity of tripartite motif protein 32. Decreasing apoptosis can be used to treat a subject having a disorder characterized by increased apoptosis, for example Limb-Girdle Muscular Dystrophy Type 2H (LGMD2H).
  • LGMD2H Limb-Girdle Muscular Dystrophy Type 2H
  • tripartite motif protein 32 protein and PIAS protein.
  • the interaction between tripartite motif protein 32 and PIAS can be increased by contacting a cell with an agent that increases an amount of tripartite motif protein 32 in the cell.
  • agents include nucleic acids that encode a protein having tripartite motif protein 32 activity, as well as specific-binding agents, such as antibodies.
  • Exemplary tripartite motif protein 32-encoding nucleic acids include, but are not limited to a nucleic acid sequence having at least 70% sequence identity to SEQ LD NOS: 1 or 12, such as at least 80%, at least 90%, at least 95%, at least 98% or even at least 99% sequence identity to SEQ LD NOS: 1 or 12.
  • a tripartite motif protein 32 nucleic acid includes SEQ ID NO: 1 or 12.
  • Agents that can be used to increase cellular activity of tripartite motif protein 32 include, but are not limited to, agents that increase the interaction between tripartite motif protein 32 and PIAS, as well as exogenous tripartite motif protein 32 supplied to the cell.
  • exogenous tripartite motif protein 32 can be supplied to the cell by an exogenous nucleic acid sequence encoding a protein having tripartite motif protein 32 activity.
  • the method includes contacting the test agent (such as one or more test agents) with a composition that includes tripartite motif protein 32 and PIAS, then determining whether the amount of binding or interaction between tripartite motif protein 32 and PIAS increased or decreased in the presence of the test agent. If the interaction is increased, this indicates that the test agent can be used to decrease apoptosis. h contrast, if the interaction is decreased, this indicates that the test agent can be used to increase apoptosis.
  • the tripartite motif protein 32 or PIAS proteins can be expressed in a cell, and the cell contacted with the test agent.
  • a cell can be transformed with one or more expression vectors encoding for tripartite motif protein 32 or PIAS, then cultured under conditions that allow expression of tripartite motif protein 32 or PIAS, assaying the transformed cell for binding between tripartite motif protein 32 or PIAS, and determining whether the test compound modulates apoptosis.
  • EXAMPLE 1 Trim32 Expression is Associated with Carcinogenesis
  • the clonal epidermal model of carcinogenesis (FIG. 1) consists of non- transformed progenitor cells and three independently initiated lineages with distinct tumor fates (Kulesz-Martin et al. Carcinogenesis, 9:171-4, 1988). Because Ha-ras mutations are rare in human squamous cell carcinomas (Field, 1996; Pierceall et al, 1991), this epidermal model lacking an activating Ha-ras gene mutation was used to identify alternative initiation and malignant conversion genes relevant to human cancer.
  • non-transformed keratinocytes (291) were treated with DMBA (7, 12-dimethylbenz[ ⁇ ] anthracene), resulting in three independently initiated clones
  • Non-transformed 291 keratinocytes exhibit characteristics of primary epidermal cultures, including regulation of proliferation and terminal differentiation by extracellular Ca 2+ , keratin patterns and cornification envelope formation indistinguishable from that of primary epidermal cultures, and lack of tumorigenicity in syngeneic newborn mice.
  • the 09C, 05C, and 03C initiated cells and the 09R tumorigenic cells were grown in high calcium Eagle's medium (HCEM), which included EMEM supplemented with 5% (v/v) fetal calf serum, 10 ng/ml EGF, 1% (v/v) antibiotic- antimycotic, and 1.4 mM CaCl 2 .
  • HCEM high calcium Eagle's medium
  • Tumorigenic 05R and 03R cells were grown in HCEM medium without EGF supplementation. All cells were cultured under identical conditions in LCEM 24 hours prior to RNA or protein harvest.
  • mice Although the tumors induced in the mice were morphologically identical to sporadic tumors induced by DMBA/TPA (7,12-dimethylbenz[ ⁇ ]anthracene/12-O- tetradecanoylphorbol-13-acetate) treatment in vivo, they lack Ha-Ras gene overexpression or mutation.
  • the cell lineages cryopreserved at different stages of transformation and tumorigenesis provide the ability to functionally test candidate oncogene activities in growth, apoptosis and in vitro transformation.
  • RNAs were extracted from cells at approximately 70% confluence using TRIzol reagent, and normal adult Balb/C mouse tissues and human tumor tissue samples were homogenized in TRIzol Reagent using a Polytron (Kinematica). RNA (10 ⁇ g) was separated on a denaturing formaldehyde agarose gel, transferred to a nylon membrane, and incubated with [ 32 P ] dCTP-labeled 1.5 Kb Trim32 probe (3 x 10 6 cpm/ml). After washing, radioactive signals were visualized by autoradiography and quantitated by phosphorimaging (Molecular Dynamics).
  • Trim32 elevation in the epidermal model was confirmed by detection of a single 3 Kb mRNA by northern blotting. All initiated (09C, 05C, and 03C) and tumorigenic (09R, 05R, and 03R) cells exhibited 2-5 fold elevated expression compared to non-transformed 291 cells (normalized to G3PDH or 7s), indicating that Trim32 mRNA is elevated at initiation and persists with tumorigenic progression and malignancy.
  • Trim32 mRNA was present in all normal mouse tissues examined by northern blotting, indicating ubiquitous expression. In normal tissues, Trim32 expression was particularly high in brain and testis, two tissues that have very low rates of apoptosis and a blood-barrier that ensures tissue integrity. The elevated expression of Trim32 protein in mouse brain was confirmed in human brain by immunoblotting with Trim32 antibody (see Example 3).
  • Mouse Trim32 cDNA was cloned and identified as the ortholog of human HT2A as follows. mRNA differential display was performed as previously described (Liu and Kulesz-Martin, Carcinogenesis, 19:683-6, 1998). The complete Trim32 cDNA was obtained by screening a normal adult mouse testis cDNA library (Stratagene) and performing ligation-anchored PCR using the Marathon cDNA Amplification Kit (Clontech) and Balb/C adult mouse brain mRNA template. The cloned mouse Trim32 cDNA sequence (SEQ LD NO: 1 GenBank AF347694) comprises a 1968 nucleotide open reading frame encoding a 655 amino acid protein (Genbank Accession No.
  • NM_053084 SEQ ID NO: 2 and is over 96% identical to human TRLM32 (SEQ LD NO: 3) in deduced amino acid sequence (FIG. 2).
  • Mouse Trim32 like human TRLM32, contains a RING domain (amino acids 20- 65 of SEQ ID NO: 2) differing from the human sequence by 1 amino acid), a B-box (amino acids 101-134 of SEQ LD NO: 2), a coiled-coil domain characteristic of the tripartite motif (TRIM) family (amino acids 136-255 of SEQ ID NO: 2), and a carboxy-terminal NHL domain (amino acids 363-655 of SEQ ID NO: 2).
  • TAM tripartite motif
  • the NHL domain in human TRTM32 is responsible for TAT protein interaction and is mutated in LGMD2H from aspartic acid to asparagine at amino acid 487. Trim32 sequencing at the genomic level indicated that both the 291 non- transformed and 03R SCC cells had wild type Trim32, verifying association of transformation-related changes with overexpression of wild type protein.
  • This example describes methods used to measure Trim32 protein and mRNA expression in mouse tumor cells. Similar methods can be used to measure expression of tripartite motif protein 32 from any organism.
  • Trim32 cDNA was cloned into pGEX (Pharmacia), and an N-terminal GST- Trim32 fusion protein was produced in bacteria and purified as described (Albor et al, Cancer Res. 58:2091-4, 1998).
  • GST-Trim32 was injected into three female New Zealand white rabbits (RPCI Laboratory Animal Resources). Antisera specificity and titer for Trim32 antigen were tested by immunoblotting cell lysates and recombinant protein.
  • Cultured cells at 70% confluence were lysed at 4°C for 1 hour in extraction buffer (20 mM HEPES, pH 7.5, 20% glycerol, 500 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 1 mM Na 3 VO 4 , 50 mM NaF, 1 mM DTT, 0.4 mM Pefabloc, 5 ⁇ l/ml PSC protector, 1 ⁇ M leupeptin, 1 ⁇ M pepstatin, and 0.1 ⁇ M aprotinin), and lysates cleared by centrifugation at 12,000 : g for 15 minutes.
  • extraction buffer (20 mM HEPES, pH 7.5, 20% glycerol, 500 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 1 mM Na 3 VO 4 , 50 mM Na
  • Tumors and normal skin were isolated from SKH-1 hairless mice treated with UVB (9.0 kJ/m 2 cumulative dose UVB) for 26 weeks or Sencar mice treated with a sub- threshold dose of DMBA (5 ⁇ g DMBA/0.2 ml acetone) and treated with TPA (2 ⁇ g TPA/0.2 ml acetone once weekly) or mezerein (4 ⁇ g mezerein/0.2 ml acetone twice weekly) for 20 weeks.
  • Mouse tumor samples were pulverized in liquid nitrogen, and protein was isolated from TRIzol lysates according to the manufacturer's instructions. Protein was quantitated using the Bradford colorimetric assay (Bio- Rad) according to manufacturer's instructions.
  • Lysates (40 ⁇ g total protein) were resolved in SDS-PAGE, transferred onto nitrocellulose membranes (Schleicher & Schuller) and immunoblotted with Trim32 antiserum or with monoclonal antibodies for GFP (Santa Cruz Biotechnology) or Hsp70 (Stressgen). Immuno-complexes were visualized by chemilummescence and quantitated using an Epson Perfection 1650 Photo Scanner and OptiQuant (Packard) software. Fast-Green staining of total protein was used as a loading control.
  • Trim32 protein (78 kDa) levels were elevated 2-6 fold in all initiated (09C, 05C, and 03C) and tumorigenic (09R, 05R, and 03R) cells of the clonal epidermal cell model, compared to non-transformed 291 cells (normalized to loading control Hsp70).
  • Trim32 protein levels were measured in mouse tumors derived by UVB- irradiation or two-stage carcinogenesis protocols. Briefly, tumors and normal skin were isolated from SKH-1 hairless mice treated with UVB (9.0 kJ/m 2 cumulative dose UVB) for 26 weeks or Sencar mice treated with a sub-threshold dose of DMBA (5 ⁇ g DMBA/0.2 ml acetone) and treated with TPA (2 ⁇ g TPA/0.2 ml acetone once weekly) or mezerein (4 ⁇ g mezerein/0.2 ml acetone twice weekly) for 20 weeks. Mouse tumor samples were pulverized in liquid nitrogen, and protein was isolated from TRIzol lysates according to the manufacturer's instructions. Protein was quantitated using the Bradford colorimetric assay (Bio-Rad) according to manufacturer's instructions.
  • Trim32 was present in all samples examined, and all tumors (6/6) induced by UVB had elevated Trim32 protein levels, ranging from 2 to 6 times non-irradiated skin from the same mouse. Two samples of non-tumorous UVB-irradiated skin taken from the back of these mice showed elevation of Trim32 expression, indicating that UNB-initiated skin may aheady have elevated Trim32 expression. A single treatment with 1500 J/m2 UVB failed to increase Trim32 expression in mouse skin up to 8 days after irradiation, ruling out the possibility that elevated Trim32 expression seen in tumors was an acute keratinocyte response to UVB irradiation.
  • qPCR quantitative real-time PCR
  • Gene expression data were collected using the 7900HT thermocycler (Applied Biosystems, Inc.) and gene-specific primers for human TRIM32 (TGTCCCTTTTGCAGCAAGATT; SEQ JD NO: 4 and GATCTTTAGCACTGTCAGATTGTCTGT; SEQIDNO: 5); 18S (CGGCTACCACATCCAAGGAA; SEQ EDNO: 6 and
  • tripartite motif protein 32 expression can be elevated early, prior to malignancy, and maintained or further increased in malignant tumors.
  • This example describes an in vitro transformation assay (Kulesz-Martin et al, Carcinogenesis, 6:1245-54, 1985) used to demonstrate that Trim32 is sufficient for epidermal cell transformation.
  • This assay based on altered response to extracellular Ca 2+ , measures an early step in epidermal cell transformation in response to a variety of chemical, physical, or viral oncogenic factors applied in vitro or in vivo. The ability to maintain colonies under conditions that induce terminal differentiation in vitro correlates with initiation, whether carcinogen is applied in vitro or in vivo.
  • the in vitro transformation assay (summarized in FIG. 4A) was applied to 291 cells retrovirally transduced with GFP, Trim32, ⁇ -sense Trim32, or activated Ha-Ras, and selected with G418 as follows.
  • the in vitro transformation assay is based on altered response to extracellular calcium ion (Ca 2+ ). Non-transformed 291 keratinocytes proliferate in culture media with 0.04 mM extracellular Ca 2+ supplemented with EGF and fibroblast conditioned media.
  • non-transformed keratinocytes When the extracellular Ca 2+ concentration is elevated (>1 mM), and EGF and conditioned media are removed, non-transformed keratinocytes accumulate differentiation-specific keratins, terminally differentiate and slough from the culture dish, while transformed keratinocytes continue to proliferate.
  • 291 cells have a spontaneous transformation frequency of O.001, indicating that the background of this assay is very low (Kulesz-Martin et al, Carcinogenesis 6:1245-54, 1985).
  • %PE [number of colonies grown at 0.04 mM Ca 2+ /number of viable cells plated] x 100).
  • HCEM media with 100 ⁇ g/ml G418 was added and exchanged twice weekly.
  • %TF [number colonies grown at 1.4 mM Ca 2+ /number of colonies grown at 0.04 mM Ca 2+ ] x 100).
  • mice were grown in LCEM with G418 (100 ⁇ g/ml), and engrafted to the skin biopsy sites of athymic nu/nu mice using an established skin-grafting technique (Kulesz-Martin et al, Carcinogenesis 9:171-4, 1988). 5 x 10 6 cells were placed on each graft site. In some mice, two weeks after grafting, TPA was applied topically once per week for 20 weeks (16 nmol or 2 ⁇ g TPA/0.2 ml acetone) to the backs of mice. Mice were euthanized when a tumor reached 1 cm in diameter.
  • Samples of tumor and uninvolved skin were placed in formalin for histopathological analysis and snap-frozen for biochemical analysis (genotyping of p53 status and immunoblotting for Trim32 and GFP protein). Some samples were analyzed by microcoscopy of hematoxylin and eosin stained sections of skin biopsies taken at 26 weeks (5 weeks after final TPA treatment).
  • Trim32:TPA-treated mice (3/6) exhibited thickened skin compared to GFP:TPA-treated mice or Trim32: solvent control.
  • a biopsy of Trim32:TPA mice revealed spongiosis (edema) and thickened epidermis with occasional dyskeratotic keratinocytes reminiscent of Bowen's Disease, a squamous cell carcinoma in situ found on sun exposed skin. Few mitotic figures were present, indicating that epidermal thickening was not due to increased proliferation.
  • Phenotypic abnormalities were absent in GFP:TPA and Trim32:solvent control mice.
  • the epidermal thickening observed in the Trim32:TPA mice persisted 12 months post-grafting (5 months after the last TPA treatment), but was seen in only a portion of the grafts and was not associated with enhanced proliferative features or papilloma formation, indicating that additional carcinogenic events or cofactors are required.
  • elevated Trim32 expression was combined with a malignant conversion-associated defect, loss of p53 function.
  • the ⁇ 53 gene is mutated in over 50% of human cancers and p53 function is altered at malignant conversion in the clonal epidermal model (Han and Kulesz-Martin, Cancer Res.
  • Trim32-expressing cells Prior to implantation, p53 genotype and Trim32 and GFP protein levels were verified. Trim32-expressing cells had 2.5 - 4 fold elevated Trim32 protein compared to their respective GFP- expressing control cell lines. All cell strains were keratinocytes, as indicated by keratin 14 detection by immunoblotting. Dishes expressing the same virus were pooled and maintained in LCEM with
  • RNA expression levels were determined by northern blotting (Trim32, GFP, and activated Ha-Ras), genotype of p53 defective cells was tested by PCR, and Trim32, GFP, and p53 protein expression levels, were determined by immunoblotting as described in the examples above. Tumors formed in p53 -/- Trim32 grafts (2 tumors/24 graft sites) beginning at 12 weeks compared to p53 -/- GFP grafts (0/24). When combined with mutant p53, tumors formed on 25 - 33% of 24 graft sites, with no statistically significant differences between p53 -/R172H Trim32 or p53 -/R172H GFP groups.
  • Histopathology of the annular plaque revealed compacted collagen bundling (collagen similar to scar tissue or the graft site) compared to uninvolved skin and an increased number and length of hair follicles compared to uninvolved skin.
  • Hair follicle density amplification within the annular plaque may be a precursor to tumorigenesis. Hair cycling in mouse skin occurs in a wave pattern with interactive signaling between neighboring follicles with 10% of the follicles in anagen, the proliferative phase, and 90% in telogen, the resting phase.
  • annular plaque phenotype has not been observed previously in hundreds of grafts of cultured cells or skin. Genotyping of tumors confirmed expression of the p53 mutant or null alleles, while the null allele was absent in the annular plaque (determined by PCR specific for the null cassette), indicating that tumors arose from the engrafted epidermal cells, whereas annular plaques were comprised of host cells.
  • This example describes methods used to determine the response of keratinocytes to inducers of apoptosis, and to demonstrate that tripartite motif protein 32 enhances cellular survival of cells exposed to UVB.
  • the common phenotype of Trim32 cells in vivo was thickening of skin, due to epidermal hyperplasia or increased number of hair follicles. This was not associated with increases in mitotic figures in vivo or cellular proliferation rates in vitro.
  • UVB was used because of its role in inducing apoptosis in normal epidermis and its significance in human skin cancer, and because of the observed elevated expression of Trim32 in UVB-induced mouse skin tumors.
  • Apoptosis underlies the sunburn reaction, a mechanism that eliminates keratinocytes with irreparable UV- induced damage (Zhuang et al, J. Interf. Cytok. Res. 20:445-54, 2000).
  • TNF ⁇ is released by skin keratinocytes upon UVB-irradiation, enhancing its apoptotic effects, and is a key mediator of sunburn (Zhuang et al, J. Immunol, 162:1440-7, 1999).
  • 291-Trim32 and 291 -GFP cells were treated in vitro with TNF ⁇ /UVB and examined for apoptosis. Apoptotic and non-apoptotic cells were distinguished in vitro by phase contrast microscopy, DNA and mitochondrial fluorescence staining, or caspase-3 activation. Briefly, 291- Trim32 or 291-GFP cells were treated at 50% confluence with 5 ng/ml mouse TNF ⁇ (R&D Systems) and/or 230 J/m 2 UVB (using 2 Westinghouse FS20T12 sun lamps with maximum emission at 310 nm) alone or in combination.
  • cytoplasmic lysates were prepared by freeze thawing of the cell pellets in hypotonic buffer (Rathbun et al, Blood, 96:4204-11 , 2000) 4.5 hours after treatment with TNF ⁇ /UNB. Reactions were performed with 30 ⁇ g cytosolic protein extract in 230 ⁇ l buffer containing 100 mM HEPES, pH 7.5, 20% glycerol, 0.1% CHAPS, 10 mM DTT, 0.1 mg/ml BSA, and 200 ⁇ M Ac-DEVD- ⁇ A (a colorimetric substrate for active caspase-3).
  • mice were anaesthetized and irradiated with 600 J/m 2 , 1200 J/m 2 , or 1800 J/m 2 (as described above) and euthanized 24 hours later. Grafts were harvested and placed in formalin for histopathological analysis. Sunburn cells were counted per number of basal cells in 4 serial sections. Apoptosis was confirmed by in situ oligo ligation (ISOL) (Seralogicals Corporation), which measures duplex blunt end DNA, a hallmark of apoptosis.
  • ISOL in situ oligo ligation
  • 291-Trim32 cells were 77% less sensitive to TNF ⁇ /UVB treatment than 291- GFP cells (FIG. 6A). Furthermore, 291-Trim32 cells exhibited 2 to 3 fold reduction in caspase-3 activity after TNF ⁇ /UVB treatment (FIG. 6A, inset). Non-apoptotic cells have faint blue Hoechst nuclear fluorescence and intense red cytoplasmic mitotracker fluorescence, while apoptotic cells have intense Hoechst fluorescence and faint cytoplasmic red mitotracker fluorescence. The percentage of apoptotic cells decreased from 65% to 15% in 291-Trim32 cells as compared to cells not expressing exogenous Trim32.
  • the apoptotic response of 291 -GFP cells was equal to that of the parental 291 cells, indicating that cell line generation alone did not alter apoptotic potential.
  • the response of Trim32-transduced cells to UVB-irradiation in vivo was measured to further illustrate this effect.
  • Apoptotic cells in the epidermis called sunburn cells (SBCs) are distinguished by their condensed, pycknotic nuclei and shrunken, eosinophihc cytoplasm (Ziegler et al, Nature 372:773-6, 1994).
  • Trim32 grafts were 2 to 2.6 fold less sensitive to apoptosis than GFP grafts irradiated with 600 J/m 2 UVB (p ⁇ 0.02, Wilcoxon rank sum test) based on fewer SBCs in the Trim32 grafts.
  • SBCs in GFP grafts increased 10 to 20 times with increasing UVB doses of 600 and 1200 J/m , respectively, with
  • TNF ⁇ is secreted by keratinocytes in response to UVB-irradiation, and the TNF ⁇ pathway is required for efficient UVB-induced apoptosis of skin in vivo (Zhuang et al, J. Immuno. 162:1440-7, 1999).
  • tripartite motif protein 32 confers cellular survival by dampening the apoptotic cellular response to TNF ⁇ after UVB induced damage, expanding the pool of target cells for further oncogenic events.
  • EXAMPLE 7 Trim32 E3-ubiquitin ligase Activity Increases After TNF ⁇ /UVB Treatment
  • Protein ubiquitylation is a fundamental process in eukaryotic cells, controlling the degradation cellular proteins through the proteosome. The process is catalyzed by the sequential action of three essential enzymes, the last of them, E3- ubiquitin ligase, providing substrate specificity.
  • E3-ubiquitin ligase A large number of E3 -ligases have been described, which can be structurally classified depending on the type of E2 interacting domain present in the protein (namely HECT domain, RING domain or U-box).
  • the RING domain is a zinc finger motif present in a variety of otherwise structurally unrelated proteins functioning as E3-ligases.
  • tripartite motif protein 32 the RING domain is part of the RBCC (RingB-Box/Coiled-coil) tripartite motif that defines the TRIM family of proteins.
  • the RING domain of tripartite motif protein 32 is consistent with tripartite motif protein 32 functioning as an E3- ubiqutin ligase.
  • tripartite motif protein 32 has E3-ubiquitin ligase activity in keratinocytes, the ubiquitylation state of Trim32 and its interaction with ubiquitylated proteins with or without TNF ⁇ /UVB treatment was determined as follows. 291 cells were transfected with myc-tagged ubiquitin (provided by Dr.
  • Transfected cells were treated with TNF ⁇ /UVB (as described in Example 6), and protein extracts were prepared 4.5 hours after treatment.
  • the proteosome inhibitor MG132 (20 ⁇ M) was added to the culture medium 2.5 hours prior to protein extraction.
  • Cells were lysed in a buffer containing 50 mM HEPES, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, and 20% glycerol with 1 ⁇ M leupeptin, 1 ⁇ M pepstatin, and 1 mM PMSF. Protein concentration was determined by the Bradford colorimetric assay.
  • Lysates were diluted to a final protein concentration of 1 ⁇ g/ ⁇ l, and a total of 400 ⁇ g of lysate protein was incubated with a 1/100 dilution of the myc-epitope specific 9E10 ascites fluid (Sigma-Aldrich) or 2 ⁇ g of the GFP-specific monoclonal antibody B-2 (Santa Cruz Biotechnology). Samples were incubated overnight at
  • Treatment with TNF ⁇ /UNB had no effect on GFP or GFP-Trim32 protein levels compared to untreated lysates.
  • a myc-specific antibody that recognizes transfected, myc-tagged ubiquitin
  • immunoblotting with a GFP-specific antibody several bands corresponding to ubiquitylated GFP-Trim32 proteins were detected, and intensity of these bands increased at least 3-fold after T ⁇ F ⁇ /UNB treatment.
  • E3-ubiquitin ligase and that tripartite motif protein 32-associated ubiquitylation is stimulated by T ⁇ F ⁇ /UVB treatment.
  • tripartite motif protein 32 expression is frequently elevated in mouse skin tumors induced by UV irradiation, and tripartite motif protein 32 expression protects keratinocytes from apoptosis induced by UVB and T ⁇ F ⁇ treatment in vitro and in vivo.
  • the results in Example 7 indicate that tripartite motif protein 32 functions as an E3-ligase. Based on this information, a yeast two-hybrid assay using full length Trim32 as bait and a mouse cDNA library from testicle as prey (where Trim32 is expressed at high levels) was used to identify tripartite motif protein 32 binding proteins.
  • tripartite motif protein 32-binding proteins could be used as substrates for tripartite motif protein 32 E3 activity.
  • Trim32 cDNA was inserted into yeast GAL4 DNA binding domain fusion expression plasmid pGBTKT7. The resulting plasmid was introduced into haploid yeast strain AH109. A strain of AH109 expressing GAL4-Trim32/TRIM32 fusion protein was selected, and mated with a transactivation domain-mouse cDNA fusion yeast library. Clones expressing TRLM32/Trim32 interacting proteins were isolated using metabolic selection markers.
  • FIG. 7A The screen isolated an almost complete cDNA sequence for a mouse Pias ⁇ gene, lacking only the first four amino acids on the amino terminus (FIG. 7A).
  • Mouse Pias ⁇ is highly homologous to the human gene PIASy (88 % identity in protein sequence; see SEQ ID NOS: 10 and 11), and comparison of the genomic sequences surrounding human PIASy and mouse Pias ⁇ genes reveals that both genes are present in orthogous chromosomal regions, surrounded by the same genes (FIG. 7B). Therefore, Pias ⁇ is the mouse ortholog of human PIASy.
  • Trim32 and Pias ⁇ proteins in yeast were specific, each protein in isolation lacking auto activation. Trim32 mutational analysis of binding revealed a complex among the protein domains. Expression plasmids for GAL4 DNA binding domain fusion to tripartite motif protein 32 protein variants and for the transactivation domain fusion to PIAS were cotransformed into yeast strain AH109. Positive clones were isolated using metabolic selection.
  • tripartite motif protein 32 variants of were examined: substitution at residue 21 of SEQ ID NO: 2, cysteine to serine; substitution at residue 489 SEQ ID NO: 2, aspartic to asparagine; deletion of RING domain, residues 1 to 102 of SEQ ID NO: 2 deleted; deletion of NHL domain, residues 361 to 653 of SEQ ID NO: 2 deleted; and deletion of coiled-coil domain, residues 143 to 318 of SEQ ID NO: 2 deleted.
  • TRIM32 for example SEQ ID NO: 3
  • Complete removal of the RING domain of Trim32 resulted in an enhancement of interaction (FIG.
  • D487N is associated with limb-girdle muscular dystrophy type 2H (LGMD2H), a slow progressing muscle wasting disease (Frosk et al, Am. J. Hum. Genet. 70:663- 72, 2002).
  • LGMD2H limb-girdle muscular dystrophy type 2H
  • a slow progressing muscle wasting disease Frosk et al, Am. J. Hum. Genet. 70:663- 72, 2002.
  • the binding assays described above were used.
  • reproduction of the D to N mutation within the NHL domain found in LGMD2H (D489N in mouse, corresponding to D487N in human) abolished binding to the Pias ⁇ , as did complete deletion of the NHL domain (FIG. 7A).
  • Example 7 increased co-immunoprecipitation with ubiquitylated proteins and ubiquitylation of Trim32 after treatment with TNF ⁇ and UVB light was observed.
  • PIAS is ubiquitylated in a tripartite motif protein 32- dependent manner
  • mouse keratinocyte cells were simultaneously transfected with plasmids for expression of GFP tagged Trim32, 6xHis tagged Pias ⁇ and myc epitope tagged ubiquitin.
  • the activity of wild type Trim32 was compared to the activity of missense mutants at the conserved Cys 21 residue in the RING domain (C21S, GFP-T32C21S) and at conserved Asp 489 residue in the NHL domain mutated in LGMD2H (D489N, GFP-LGMD2H).
  • Cells were also treated with UVB and TNF ⁇ , using the methods described in the above examples.
  • Ubiquitylated proteins were immunoprecipitated with a myc specific antibody, and ubiquitylated Pias ⁇ detected by immunoblotting using the methods described in the above examples.
  • the RING domain is essential for the function of RING containing E3 -ubiquitin ligases, and failure of both GFP-T32C21 S and GFP-Trim32DRING to promote Pias ⁇ ubiquitylation supports the role of as an E3-ubiquitin ligase targeting Pias ⁇ for ubiquitylation.
  • the LGMD2H mutant form of Trim32 failed to stimulate Pias ⁇ ubiquitylation beyond the levels found in non-transfected cells.
  • PIAS Protein ubiquitylation typically leads to degradation through the proteosome pathway.
  • PIAS should be less stable in the presence of tripartite motif protein 32 than in its absence or in the presence of mutants T32C2 IS or T32LGMD2H.
  • mouse keratinocytes were transfected with expression vectors for GFP-Pias ⁇ and the Trim32 forms GFP-Trim32, GFP-T32C21S and GFP- T32LGMD2H. New protein synthesis was blocked with cycloheximide (25 ⁇ M), and cells were then treated with UVB plus TNF ⁇ using the methods described above.
  • the levels of Pias ⁇ protein decrease with time after cycloheximide block and UVB/TNF ⁇ treatment in cells co-transfected with wild type Trim32, but not in cells transfected with the RING domain and LGMDH2 mutants.
  • tripartite motif protein 32 promoted the ubiquitylation and degradation of PIAS upon stimulation with UVB light and TNF ⁇ , but only with an intact RING domain was present in tripartite motif protein 32.
  • EXAMPLE 12 Elevation of PIASy in limb-girdle muscle dystrophy type 2H Subjects Patients with limb-girdle muscle dystrophy type 2H are homozygous for the
  • Duchenne muscle dystrophy patients (who carry a mutation on the dystrophyn gene) as well as from healthy donors, and the levels of PIASy determined by immunoblotting both in control and UVB/TNF ⁇ treated cells, using the methods disclosed herein.
  • PIASy protein displayed increased stability in fibroblasts isolated from an LGMD2H patient, compared to a healthy donor, which could be restored to normal by ectopic expression of wild type tripartite motif protein 32.
  • Trim32 controls Pias ⁇ stability through promotion of ubiquitylation in response to UVB/TNF ⁇ treatment, and the dependence on the RING domain indictes that Trim32 mediates this effect as an E3- ubiquitin ligase. Trim32 has been reported as a cytoplasmic protein, concentrated in speckles
  • PIAS proteins are generally considered nuclear due to their function as SUMO ligases, although the Drosophila PIAS protein Su(var)2-10, the only known Drosophila PIAS family member, is present during interphase both in the nucleus and the cytoplasm, with higher concentrations near the nuclear periphery (Wang et al, Carcinogenesis 23:635-43, 2002).
  • Trim32/Pias ⁇ interaction was determined.
  • GFP-Pias ⁇ appears both nuclear and with diffuse cytoplasmic staining, and GFP-Trim32 is speckled cytoplasmic with some likely nuclear speckles.
  • EXAMPLE 14 Anti-Apoptoic Activity of Trim32 Due to its Ubiquitylation of Pias ⁇
  • Trim32 protein has a protective effect on keratinocyte apoptosis induced by exposure to UVB and TNF ⁇ (see Example 6).
  • the induction of apoptosis by UVB/TNF ⁇ treatment in mouse keratinocyte cells co-expressing ectopic Pias ⁇ and wild type and mutant forms of Trim32 was determined.
  • Cells were co-transfected with plasmids for the expression of GFP tagged Trim32/TRLM32, 6x histidine tagged PIASy/Pias ⁇ , and myc tagged ubiquitin.
  • ubiquitylated proteins were precipitated with a myc specific antibody, and the presence of ubiquitylated PIASy/Pias ⁇ detected by immunoblotting with a 6x histidine specific antibody.
  • EXAMPLE 15 Method of Screening for Therapeutic Agents This example provides methods that can be used to screen test agents for an ability to modulate apoptosis. The method involves determining whether a test agent modulates binding between tripartite motif protein 32 and PIAS.
  • a test agent, or more than one test agents, are exposed to a composition that includes tripartite motif protein 32, such as human TRTM32 or mouse Trim32, and PIAS, such as human PIASy or mouse Pias ⁇ proteins. Subsequently, the amount of binding or interaction between tripartite motif protein 32 and PIAS is determined, for example by immunoprecipitation followed by immunoblotting as described above. If the interaction between the proteins is increased, this indicates that the test agent can be used to decrease apoptosis. In contrast, if the interaction is decreased, this indicates that the test agent can be used to increase apoptosis. Tripartite motif protein 32 and PIAS proteins can be expressed in a cell, and the cell contacted with the test agent.
  • tripartite motif protein 32 such as human TRTM32 or mouse Trim32
  • PIAS such as human PIASy or mouse Pias ⁇ proteins.
  • a cell can be transformed with one or more expression vectors encoding for tripartite motif protein 32 or PIAS, then cultured under conditions that allow expression of tripartite motif protein 32 or PIAS, assaying the transformed cell for binding between tripartite motif protein 32 and PIAS, and determining whether the test compound modulates apoptosis.
  • EXAMPLE 16 Disruption of Gene Expression
  • This example describes methods that can be used to disrupt expression of tripartite motif protein 32 to increase apoptosis, or to disrupt expression of PIAS to decrease apoptosis. Such methods are useful when it is desired to modulate apoptosis.
  • disrupted expression of tripartite motif protein 32 is used to increase apoptosis in subjects suffering from cancer, such as a skin cancer or leukemia, or atopic dermatitis.
  • disrupted expression of PIAS is used to decrease apoptosis in subjects suffering from muscular dystrophy, such as LGMD2H.
  • One approach to disrupting tripartite motif protein 32 or PIAS function or expression is to use antisense oligonucleotides.
  • a desired mRNA sequence is examined, such as tripartite motif protein 32 or PIAS mRNA sequence. Regions of the sequence containing multiple repeats, such as TTTTTTTT, are not as desirable because they will lack specificity. Several different regions can be chosen. Of those, oligos are selected by the following characteristics: those having the best conformation in solution; those optimized for hybridization characteristics; and those having less potential to form secondary structures. Antisense molecules having a propensity to generate secondary structures are less desirable.
  • Plasmids including antisense sequences can be generated. For example, cDNA fragments or variants coding for a desired protein, such as tripartite motif protein 32 or PIAS are PCR amplified. The nucleotides can be amplified using Pfu DNA polymerase (Sfratagene) and cloned in antisense orientation a vector, such as pcDNA vectors (InVitrogen, Carlsbad, CA). The nucleotide sequence and orientation of the insert can be confirmed by sequencing using a Sequenase kit (Amersham Pharmacia Biotech).
  • antisense refers to a nucleic acid capable of hybridizing to a portion of an RNA molecules (such as mRNA) by virtue of some sequence complementarity.
  • the antisense nucleic acids disclosed herein can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell or a subject, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • Antisense nucleic acids are polynucleotides, and can be oligonucleotides
  • a tripartite motif protein 32 or PIAS antisense polynucleotide recognizes any species of tripartite motif protein 32 or PIAS, respectively.
  • the oligonucleotide is at least 10, 15, or 100 nucleotides, or a polynucleotide of at least 200 nucleotides.
  • antisense nucleic acids can be much longer.
  • the nucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, and can include other appending groups such as peptides, or agents facilitating transport across the cell membrane (Letsinger et al, Proc. Natl. Acad.
  • a tripartite motif protein 32 or PIAS antisense polynucleotide can be modified at any position on its structure with substituents generally known in the art.
  • a modified base moiety can be 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N ⁇ 6-sopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine,
  • the antisense polynucleotide disclosed herein can include at least one modified sugar moiety such as arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.
  • modified sugar moiety such as arabinose, 2-fluoroarabinose, xylose, and hexose
  • a modified component of the phosphate backbone such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a form
  • an antisense polynucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, Nucl Acids Res. 15:6625-41, 1987).
  • the oligonucleotide can be conjugated to another molecule, such as a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization- triggered cleavage agent.
  • Oligonucleotides can include a targeting moiety that enhances uptake of the molecule by cells.
  • the targeting moiety can be a specific binding molecule, such as an antibody or fragment thereof that recognizes a molecule present on the surface of the cell, such as a hair follicle cell.
  • catalytic nucleic acid compounds such as ribozymes or anti-sense conjugates
  • Ribozymes can be synthesized and administered to the subject, or can be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (as in WO 9523225, and Beigelman et al. Nucl. Acids Res. 1995, 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of antisense with a metal complex, such as terpyridylCu (11), capable of mediating mRNA hydrolysis, are described in Bashkin et al.
  • Polynucleotides disclosed herein can be synthesized by standard methods, for example by use of an automated DNA synthesizer.
  • phosphorothioate oligos can be synthesized by the method of Stein et al. (Nucl. Acids Res. 1998, 16:3209)
  • methylphosphonate oligos can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. Natl. Acad. Sci. USA 85:7448-51, 1988).
  • a tripartite motif protein 32 or PIAS antisense oligonucleotide includes catalytic RNA, or a ribozyme (see WO 90/11364, Sarver et al, Science 247:1222-5, 1990).
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al, Nucl Acids Res. 15:6131-48, 1987), or a chimeric RNA-DNA analogue (Inoue et al. , FEBS Lett. 215 :327-30, 1987).
  • the antisense polynucleic acids disclosed herein include a sequence complementary to at least a portion of an RNA transcript of a tripartite motif protein 32 or PIAS gene.
  • a sequence can be complementary to at least a portion of an RNA, meaning a sequence having sufficient complementarily to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded tripartite motif protein 32 or PIAS antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation can be assayed.
  • the ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid.
  • the longer the hybridizing nucleic acid the more base mismatches with a tripartite motif protein 32 or PIAS RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • the relative ability of polynucleotides (such as oligonucleotides) to bind to complementary strands is compared by determining the T m of a hybridization complex of the poly/oligonucleotide and its complementary strand. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity).
  • a reduction in UV absorption indicates a higher T m .
  • the higher the T m the greater the strength of the binding of the hybridized strands.
  • As close to optimal fidelity of base pairing as possible achieves optimal hybridization of a poly/oligonucleotide to its target RNA.
  • the amount of antisense nucleic acid which is effective in the treatment of a particular disease or condition depends on the nature of the disease or condition, and can be determined by standard clinical techniques. For example, it can be useful to use compositions to achieve sustained release of an antisense nucleic acid. In another example, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable cells of the skin, such as fibroblast or keratinocyte antigens (Leonetti et al. Proc. Natl. Acad. Sci. USA 1990, 87:2448-51; Renneisen et al. J. Biol. Chem. 1990, 265:16337-42).
  • Tripartite motif protein 32 or PIAS antisense oligonucleotides can therefore be used to disrupt cellular expression of a tripartite motif protein 32 or PIAS protein.
  • a subject suffering from a disease or condition in which increased apoptosis of cells is desired can be treated with a therapeutically effective amount of tripartite motif protein 32 antisense molecule.
  • the tripartite motif protein 32 antisense After the tripartite motif protein 32 antisense has produced an effect (tripartite motif protein 32 levels are downregulated), for example after 24-48 hours, the subject can be monitored for increased apoptosis using the methods disclosed herein.
  • a subject suffering from a disease or condition in which decreased apoptosis of cells is desired for example myoblast cells in a subject having LGMD2H
  • the subject can be monitored for decreased apoptosis using the methods disclosed herein.
  • the treatments disclosed herein can also be used prophylactically, for example to inhibit or prevent progression to of a disorder such as UV damage to skin cells in which increased apoptosis is desired, or a disorder such as LGMD2H in which decreased apoptosis is desired.
  • a disorder such as UV damage to skin cells in which increased apoptosis is desired, or a disorder such as LGMD2H in which decreased apoptosis is desired.
  • Such administration is indicated where the treatment is shown to have utility for treatment or prevention of the disorder.
  • Prophylactic use is indicated in conditions known or suspected of progressing to disorders (such as cancer) associated with increased or decreased amounts of apoptosis.
  • Various delivery systems for administering the therapies disclosed herein are known, and include encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (Wu and Wu, J. Biol. Chem. 1987, 262:4429-32), and construction of therapeutic nucleic acids as part of a retro viral or other vector.
  • Methods of introduction include, but are not limited to, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal, vaginal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • compositions disclosed herein are delivered locally to the area in need of treatment, for example by topical application.
  • administration can be by direct administration at a site where increased or decreased apoptosis is desired.
  • liposomes are used as a delivery vehicle. Liposomes fuse with the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the target cells for a sufficient time for fusion to occur, using various means to maintain contact, such as isolation and binding agents.
  • Liposomes can be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus.
  • the lipids may be any useful combination of known liposome forming lipids, including cationic lipids, such as phosphatidylcholine. Other potential lipids include neutral lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
  • the procedure described by Kato et al. J. Biol Chem. 1991, 266:3361
  • Kato et al. J. Biol Chem. 1991, 266:3361
  • compositions which include a therapeutically effective amount of a tripartite motif protein 32 or PIAS protein, RNA, DNA, antisense molecule, ribozyme, siRNA, or specific-binding agent, alone or with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions or methods of treatment can be administered in combination with other therapeutic treatments, such as other agents that reduce the number of cancer cells (for example chemotherapy or radiation therapy) or other agents used to treat muscular dystrophy, such as corticosteroids.
  • compositions and formulations suitable for pharmaceutical delivery of the DNA, RNA, proteins, and specific-binding agents herein disclosed hi general, the nature of the carrier will depend on the mode of administration being employed.
  • parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, sesame oil, glycerol, ethanol, combinations thereof, or the like, as a vehicle.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, sodium saccharine, cellulose, magnesium carbonate, or magnesium stearate.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Embodiments of the disclosure including medicaments can be prepared with conventional pharmaceutically acceptable carriers, adjuvants and counterions as would be known to those of skill in the art.
  • tripartite motif protein 32 or PIAS protein, RNA, DNA, antisense molecule, siRNA, ribozyme, or specific-binding agent effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays can be employed to identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Instructions for use of the composition can also be included.
  • compositions of tripartite motif protein 32 or PIAS peptides for example a composition that includes at least 50%, for example at least 90%, of a peptide or variant, fragment, or fusion thereof.
  • Such compositions are useful as therapeutic agents when constituted as pharmaceutical compositions with the appropriate carriers or diluents.
  • nucleic acid is delivered intracellularly (for example by expression from a nucleic acid vector or by receptor-mediated mechanisms).
  • the therapeutic molecule is a nucleic acid or antisense molecule
  • administration can be achieved by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, for example by use of a retroviral vector (see U.S. Patent No.
  • nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • the vector pcDNA is an example of a method of introducing the foreign cDNA into a cell under the control of a strong viral promoter (CMV) to drive the expression.
  • CMV viral promoter
  • retroviral vectors such as pRETRO-ON, Clontech
  • pRETRO-ON pRETRO-ON, Clontech
  • the present disclosure includes all forms of nucleic acid delivery, including synthetic oligos, naked DNA, plasmid and viral, integrated into the genome or not.
  • Administration of Antibodies include all forms of nucleic acid delivery, including synthetic oligos, naked DNA, plasmid and viral, integrated into the genome or not.
  • the therapeutic molecule is a specific-binding agent, such as an antibody that recognizes a tripartite motif protein 32 or PIAS protein
  • administration can be achieved by direct topical administration or injection, or by use of microparticle bombardment (for example, a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents. Similar methods can be used to administer proteins or variants thereof.

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Abstract

L'activation de protéine à motif tripartite 32 favorise la carcinogenèse en bloquant certains trajets de signalisation apoptotique induits par le stress, tandis que l'inactivation de la signalisation de cette protéine exacerbe la signalisation apoptotique dans la dystrophie musculaire. De plus, vis-à-vis des protéines PIAS, qui sont des inhibiteurs de transducteur de signal activé et d'activateur de transcription, ladite protéine est capable d'interaction, de liaison et d'ubiquitylation, pour favoriser la dégradation de ces PIAS. Une interaction de ladite protéine et des PIAS établit un mécanisme permettant de modifier les réponses apoptotiques pour le traitement de différents troubles. L'invention concerne à la fois des procédés relatifs à la modulation de l'apoptose (par exemple, augmentation ou diminution de celle-ci) et des procédés d'analyse visant à identifier des agents qui modulent l'apoptose.
PCT/US2003/033599 2002-10-22 2003-10-22 Regulation de reponse apoptotique par interaction de proteine a motif tripartite 32 et d'inhibiteurs pias Ceased WO2004038373A2 (fr)

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JP2010539914A (ja) * 2007-09-28 2010-12-24 イーエムベーアー−インスティトゥート・フューア・モレクラレ・ビオテヒノロギー・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング 幹細胞および前駆細胞の増殖および分化能力の調節方法

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AU2002349784A1 (en) * 2001-12-03 2003-06-17 Asahi Kasei Pharma Corporation Nf-kappab activating genes

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* Cited by examiner, † Cited by third party
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JP2010539914A (ja) * 2007-09-28 2010-12-24 イーエムベーアー−インスティトゥート・フューア・モレクラレ・ビオテヒノロギー・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング 幹細胞および前駆細胞の増殖および分化能力の調節方法

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