METHODS AND REAGENTS FOR INHIBITING ER STRESS-INDUCED APOPTOSIS

BACKGROUND OF THE INVENTION Aberrant regulation of apoptotic pathways is implicated in a number of human pathologies, including AIDS, autoimmune diseases, cancer, and neurodegenerative disorders. Accumulating evidence has established a major role for the Inhibitor of Apoptosis (IAP) family of proteins in the suppression of apoptosis. The IAP proteins are characterized by the baculoviral IAP repeat (BIR) domain, and to date, eight IAP family members have been identified in humans. The anti- apoptotic function of IAPs such as XIAP, HIAPl, and HIAP2 may be attributed to the ability of their BIR domains to directly bind to and inhibit caspases, members of a family of cysteine proteases responsible for inducing apoptosis. In particular, the BIR2 domains of XIAP, HIAPl, and HIAP2 bind to and inhibit the effector caspase-3 and -7, whereas the BIR3 domains of these IAPs suppress the activity of the initiator caspase-9. XIAP, HIAPl, and HIAP2 also possess a carboxy-terminal RING zinc-finger that functions as an E3 ubiquitin ligase. The intrinsic ubiquitin ligase activity of the RING domain has been shown to promote the ubiquitination and targeting of caspase-3 and -7 to the proteasomes, suggesting that degradation of caspases may be another mechanism by which the IAPs suppress apoptosis.

Despite high sequence homology, the IAPs may participate in distinct cellular pathways. For instance, XIAP is involved in the bone morphogenetic protein (BMP) signaling pathway as a positive regulator linking BMP receptors and TABl-TAKl, whereas HIAPl and HIAP2 are components of the protein complex that forms on the cytoplasmic tail of the 75 kDa tumour necrosis factor receptor 2 (TNFR2) via an association through TRAFl and TRAF2. Although HIAPl and HIAP2 are not as potent as XIAP in both in vitro caspase-3 inhibition assays and in most cell death models, HIAP2-transfected cells are disproportionately resistant to

tumor necrosis factor (TNF)-mediated cell death, further pointing to unique roles for the individual IAPs.

The endoplasmic reticulum (ER) has recently emerged as a key component in the processing of apoptotic signals. The ER serves as the major reservoir for free Ca ²⁺ ions and thereby plays a critical role in Ca ²⁺ homeostasis. While the transfer of Ca ²⁺ from the ER to the mitochondria is required for the initiation of programmed cell death by some apoptotic stimuli, other triggers solely engage the ER Ca ²⁺ gateway. In rodent cells, ER-associated caspase-12 participates in Ca ²⁺ - dependent cell death. Following ER stress, translocation of cytosolic caspase-7 to the ER surface is required for the cleavage and activation of caspase-12. In turn, activated caspase-12 propagates the apoptotic cascade by directly activating caspase-9 that subsequently cleaves and activates caspase-3 in a cytochrome c- independent manner. However, the corresponding pathway in humans does not appear to be fully conserved, in that the human caspase-12 gene has been shown to have sustained inactivating mutations encoding the crucial SHG box.

Thus, there is a need for a better understanding of the mechanism of ER stress-induced apoptosis in humans. This better understanding would allow for improved methods of treatment.

SUMMARY OF THE INVENTION

We now show that in ER stress-induced apoptosis, caspase-2 localized to the ER initiates the apoptotic cascade. Following ER stress, caspase-2 processing is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). The downregulation of caspase-2 by small interfering RNA decreases ER stress-mediated apoptosis. Among HIAPl, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Overexpression of HIAP2 suppresses ER stress-induced caspase-2 activity and apoptosis. Conversely, downregulation of HIAP2 promotes cell death triggered by ER stressors. Our results thus reveal a novel mechanism by which HIAP2 can determine cell fate in ER-initiated apoptosis by modulating the activity of caspase-

2. In view of these results, we propose that HIAP2 polypeptides and polynucleotides can be used in various drug screening and therapeutic applications. Accordingly, in a first aspect, the invention features a method for identifying a compound that inhibits apoptosis (e.g., ER stress-induced apoptosis). This method includes the steps of (a) contacting a polypeptide having a HIAP2 BIR2 or HIAP2 BIR1-BIR2 linker region (e.g., a human HIAP2 BIR2 or HIAP2 BIRl- BIR2 linker region) with a candidate compound; and (b) determining whether the candidate compound binds the polypeptide. A candidate compound that binds the polypeptide is identified as a compound that inhibits apoptosis. The polypeptide may be a full-length HIAP2 polypeptide (e.g., human HIAP2), or may be any BIR2-containing portion or HIAP2 BIR1-BIR2 linker region-containing portion thereof. The polypeptide may also be a chimeric polypeptide containing the BIR2 portion or HIAP2 BIR1-BIR2 linker of HIAP2. In one preferred embodiment, the polypeptide is human HIAP2 BIR2, with an optional N-terminal methionine. In another embodiment , the polypeptide is the human HIAP2 BIR1-BIR2 linker region, with an optional N-terminal methionine.

In another aspect, the invention features another method for identifying a compound that inhibits apoptosis (e.g., ER stress-induced apoptosis). This method includes the steps of: (a) contacting (i) a first polypeptide that includes a HIAP2 BIR2 (e.g., human HIAP2 BIR2) or HIAP2 BIR1-BIR2 linker region (e.g., human HIAP2 BIR1-BIR2 linker region), (ii) a second polypeptide that includes a HIAP2 BIR2- or HIAP2 BIR1-BIR2 linker region- interacting portion of caspase-2, caspase-3, caspase-4, caspase-7, or caspase-12, and (iii) a candidate compound; and (b) determining whether the candidate compound interferes with the interaction between the first polypeptide and the second polypeptide. A candidate compound that interferes with said interaction is identified as a compound that inhibits apoptosis.

The first polypeptide may be a full-length HIAP2 polypeptide (e.g., human HIAP2), or may be any BIR2- or HIAP2 BIR1-BIR2 linker region-containing portion thereof. The polypeptide may also be a chimeric polypeptide containing

the BIR2 portion of HIAP2 or a HIAP2 BIR1-BIR2 linker region. In one preferred embodiment, the polypeptide is human HIAP2 BIR2, with an optional N-terminal methionine. In another embodiment , the polypeptide is the human HIAP2 BIRl- BIR2 linker region, with an optional N-terminal methionine. The second polypeptide may be a full-length caspase (e.g. human caspase-

2), or may be any portion thereof that interacts with a HIAP2 BIR2 or a HIAP2 BIR1-BIR2 linker region, with an optional N-terminal methionine. The polypeptide may also be a chimeric polypeptide containing the HIAP2 BIR2- or HIAP2 BIR1-BIR2 linker region-interacting portion of a caspase. In a third aspect, the invention features a method for identifying a compound that may be useful for reducing apoptosis associated with cerebral ischemia, acute renal failure, alcoholic liver injury, diabetes, lysosomal storage disorders, and neurodegenerative diseases, such as Alzheimer' s disease, Huntington' s disease, or Parkinson' s disease. This method includes the steps of: (a) contacting a polypeptide having a HIAP2 BIR2 or HIAP2 BIR1-BIR2 linker region with a candidate compound; and (b) determining whether the candidate compound binds the polypeptide. A candidate compound that binds the polypeptide is identified as a compound that may be useful for reducing apoptosis associated with cerebral ischemia, acute renal failure, alcoholic liver injury, diabetes, a lysosomal storage disorder, or a neurodegenerative disease.

In still another aspect, the invention features a method for identifying a compound that may be useful for reducing apoptosis associated with cerebral ischemia, acute renal failure, alcoholic liver injury, diabetes, lysosomal storage disorders, and neurodegenerative diseases. This method includes the steps of: (a) contacting (i) a first polypeptide having a HIAP2 BIR2 or HIAP2 BIR1-BIR2 linker region, (ii) a second polypeptide having a HIAP2 BIR2- or HIAP2 BIRl- BIR2 linker region-interacting fragment of caspase-2, caspase-3, caspase-4, caspase-7, or caspase- 12, and (iii) a candidate compound; and (b) determining whether the candidate compound interferes with the interaction between the first polypeptide and the second polypeptide. A candidate compound that interferes

with the interaction is identified as a compound that may be useful for reducing apoptosis associated with cerebral ischemia, acute renal failure, alcoholic liver injury, diabetes, a lysosomal storage disorder, or a neurodegenerative disease. In still another aspect, the invention features a method for identifying a compound that stabilizes the interaction between (i) a first polypeptide that includes a HIAP2 BIR2 (e.g., human HIAP2 BIR2) or a HIAP2 BIR1-BIR2 linker region (e.g., the human HIAP2 BIR1-BIR2 linker region) and (ii) a second polypeptide that includes a HIAP2 BIR2- or HIAP2 BIR1-BIR2 linker region- interacting portion of caspase-2, caspase-3, caspase-4, caspase-7, or caspase-12. This method includes the steps of: (a) contacting the first polypeptide, (ii) the second polypeptide, and (iii) a candidate compound; and (b) determining whether the candidate compound stabilizes the interaction between the first polypeptide and the second polypeptide.

The first polypeptide may be a full-length HIAP2 polypeptide (e.g., human HI AP2), or may be any BIR2- or HIAP2 BIR 1 -BIR2 linker region-containing portion thereof. The polypeptide may also be a chimeric polypeptide containing the BIR2 portion of HIAP2 or the HIAP2 BIR1-BIR2 linker region. In one preferred embodiment, the polypeptide is human HIAP2 BIR2, with an optional N- terminal methionine. In another embodiment , the polypeptide is the human HIAP2 BIR1-BIR2 linker region, with an optional N-terminal methionine.

The second polypeptide may be full-length caspase-2 (e.g. human caspase- 2), or may be any portion thereof that interacts with a HIAP2 BIR2 or a HIAP2 BIR1-BIR2 linker region, with an optional N-terminal methionine. The polypeptide may also be a chimeric polypeptide containing the HIAP2 BIR2- or HIAP2 BIR1-BIR2 linker region- interacting portion of caspase-2.

In still another aspect, the invention features a method for enhancing ER stress-induced apoptosis in a patient in need thereof. This method includes the steps of administering to the patient a HIAP2 antisense compound or a HIAP2 siRNA in an amount effective to enhancing ER stress-induced apoptosis.

The HIAP2 antisense compound can be any suitable HIAP2 antisense compound. Exemplary HIAP2 antisense compounds that may be used in the method of the invention are described in U.S. Patent Publication No. 20040009599 and PCT Publication No. WO 03/080638 A2, each of which is hereby incorporated by reference. The HIAP2 siRNA can be any suitable HIAP2 siRNA.

In another aspect, the invention features a method of reducing ER stress- induced apoptosis in a patient in need thereof. This method includes the steps of administering to the patient an expression vector including a polynucleotide encoding a HIAP2 polypeptide (e.g., human HIAP2) or an apoptosis-reducing fragment thereof (e.g., one that includes a HIAP2 BIR2 or a HIAP2 BIR1-BIR2 linker region), wherein expression of the polypeptide in the patient reduces ER stress-induced apoptosis. The apoptosis may be associated, for example, with cerebral ischemia, acute renal failure, alcoholic liver injury, diabetes, a lysosomal storage disorder, or a neurodegenerative disease. By an "effective amount" is meant the amount of a compound (e.g., a nucleobase oligomer) required to ameliorate the symptoms of a disease, inhibit the growth of the target cells, reduce the size or number of tumors, inhibit the expression of an IAP, or enhance apoptosis of target cells, relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of abnormal proliferation (i.e., cancer) varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. By "enhancing apoptosis" is meant increasing the number of cells that apoptose in a given cell population (e.g., cancer cells, lymphocytes, fibroblasts, or any other cells). It will be appreciated that the degree of apoptosis enhancement provided by an apoptosis-enhancing compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis that identifies a nucleobase oligomer that enhances apoptosis

otherwise limited by an IAP. Preferably, "enhancing apoptosis" means that the increase in the number of cells undergoing apoptosis is at least 10%, more preferably the increase is 25% or even 50%, and most preferably the increase is at least one-fold, relative to cells not administered a nucleobase oligomer of the invention but otherwise treated in a substantially similar manner. Preferably the sample monitored is a sample of cells that normally undergo insufficient apoptosis (i.e., cancer cells). Methods for detecting changes in the level of apoptosis (i.e., enhancement or reduction) are described herein.

By "hybridize" is meant pair to form a duplex or double- stranded complex containing complementary paired nucleobase sequences, or portions thereof.

Preferably, hybridization occurs under physiological conditions, or under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30 ⁰ C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at

42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, more preferably of at least about 42 ⁰ C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. Typically, complementary nucleobases hybridize via hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 85%, but preferably 90%, more preferably 95%, most preferably 97%, or even 99% identity to a reference amino acid or nucleic acid sequence.

Sequence identity is typically measured using a sequence analysis program (e.g., BLAST 2; Tatusova et al., FEMS Microbiol Lett. 174:247-250, 1999) with the default parameters specified therein. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine and tyrosine. By "modulating" is meant conferring a change, either by decrease or increase, in IAP biological activity that is naturally present within a particular cell or sample. Preferably, the change in response is at least 5%, more preferably, the change in response is 20% and most preferably, the change in response level is a change of more than 50% relative to the levels observed in naturally occurring IAP biological activity.

By "substantially pure polypeptide" is meant a polypeptide or peptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the polypeptide is an IAP polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure IAP polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding an IAP polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different

from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes. By "caspase-2" is meant any naturally occurring caspase-2 polypeptide or having conservative substitutions, or any polypeptide substantially identical to a naturally occurring caspase-2 polypeptide. There are four human caspase-2 isoforms: isoform 1 (gi:39995059), isoform 2 (gi:4502575), isoform 3

(gi:39995061), and isoform 4 (gi: 14790186). By "caspase-3" is meant any naturally occurring caspase-3 polypeptide or having conservative substitutions, or any polypeptide substantially identical to a naturally occurring caspase-3 polypeptide.

By "caspase-4" is meant any naturally occurring caspase-4 polypeptide or having conservative substitutions, or any polypeptide substantially identical to a naturally occurring caspase-4 polypeptide.

By "caspase-7" is meant any naturally occurring caspase-7 polypeptide or having conservative substitutions, or any polypeptide substantially identical to a naturally occurring caspase-7 polypeptide.

By "caspase-12" is meant any naturally occurring caspase-12 polypeptide or having conservative substitutions, or any polypeptide substantially identical to a naturally occurring caspase-12 polypeptide.

By "HIAP2" is meant a naturally occurring HIAP2 polypeptide.

Exemplary HIAP2 polypeptides are human HIAP2 (gi:2497238), mouse HIAP2

(gi:2062676), rat HIAP2 (gi: 11120700), dog HIAP2 (gi:57086023), chimpanzee HIAP2(gi: 55636901), orangutan HIAP2 (gi:55729496), cow HIAP2 (gi:

61816378). HIAP2 polypeptides are also referred to as cIAP-1 and MIHB polypeptides.

By "HIAP2 BIR2" is meant a polypeptide having the following sequence:

Glu-Xaa _p0 i _ar -Xaa _small -Arg-Xaa _Hphob , _c -Xaa-Xaa _alcohol -Xaa _aromatlc -Xaal-Xaal-Tφ- Xaa _sma n-Xaa2-Xaa 1 -Xaa 1 -Xaaπphobic-Xaaaiiphahc- Xaa _a i _coho rPro-Xaa 1 -Xaa _ch a _r ged-Leu-

Ala- Xaa _po i _ar -Ala-Gly- Xaaπ _phob i _c -Tyr-Tyr-Xaal-Xaa _tmy -Xaal- Xaa _sma n-Asp- Xaa _po i _ar -Val-Xaa _A/Q -Cys-Phe-Xaal-Cys- Xaa _sma u-Gly- Xaa _po i _ar -Leu-Xaal- Xaa _sma n-

Trp- Xaa _ne g _C harged"X ^aa R/P"X ^aa l ^" Asp-Xaa _am photeπc ^' X ^aa small ^" X ^aa non-polarHphobic"X ^aa alcohol"

Glu-His- Xaa 1 - Arg-His- Xaaaromanc-Xaap/H-Xaapoiar-Cy s-Xaap/p/R-Phe-Xaaaiφhatic (SEQ ID NO: 1), wherein: Xaal = any amino acid; Xaa2 = any amino acid or is absent; Xaa _alcohol = Ser or Thr; Xaa _ahphatlc = Iso, Leu, or VaI; Xaa _amphoteπc = His, Lys, Arg, GIu, or Asp; XaaA _/Q = Ala or GIn; Xaa _aromatlc = Phe, Tyr, or Trp; Xaa _char g _ed = Asp, GIu, His, Lys, or Arg; Xaap _/ p _/R = Phe, Pro, or Arg; Xaa _Hphob i _c = Iso, Leu, VaI, Cys, Ala, GIy, Met, Phe, Tyr, Tryp, or His; Xaa _negcharged = Asp or GIu; Xaa _{non- po} i _arH p _hob i _c = Iso, Leu, VaI, Met, Phe, Tyr, or Trp; Xaa _P/H = Pro or His; Xaa _p ^ = Tyr, Trp, His Lys, Arg, glu, GIn, Asp, Asn, Ser, or Thr; Xaa _R/ p = Arg or Pro; Xaa _sma n = VaI, Cys, Ala, GIy, Asp, Asn, Ser, Thr, or Pro; and Xaa _tiny = Ala, GIy, or Ser. Human HIAP2 BIR2 is located at amino acid residues 184-250 of human HIAP2 and has the sequence of EEARFLTYHMWPLTFLSPSELARAGFYYIGPGDRVACFACGG KLSNWEPKDDAMSEHRRHFPNCPFL (SEQ ID NO: 2).

By "HIAP2 BIR1-BIR2 linker region" is meant a polypeptide having the following sequence : Xaa _Q/H -Xaa _sma i _r Leu-Xaa _a i _iphat i _c - Ser-Xaa2-Xaal -Xaa 1 -Xaa 1 - Xaal- Xaa _tmy -Xaa 1 -Xaal -Ly s-Asn-Xaal- Ser-Xaal -Xaal -Xaal -Xaal -Xaal -Xaa2- Xaa2-Xaa2- Xaa2-Phe-Xaal-His-Ser-Xaal-Xaal-Xaa2-Xaa2-Xaa2-Xaa2-Xaa2- Leu-Asp-Xaal-Xaal- Xaa2-Xaa2-Xaa2-Xaa2-Xaa2-Gly-Xaal-Xaal-Ser-Xaal- X ^aa ah _P h _at ic-X ^aa 1 -X ^aa 1 -X ^aa smair Pro-Xaa _aliphatlc -Xaa _small - Ser- Arg- Ala-Xaa _sma n- X ^aa _amphoteπc -Asp-XaaHph _ob i _c -Ser-Xaal-Xaal- Xaal-Xaa2-Xaa _smair Pro-Xaal-Ser- X ^aa _aromat i _c -Ala-Met-Ser-Thr (SEQ ID NO: 3), wherein Xaal = any amino acid, Xaa2 = any amino acid or is absent, Xaa _a i, _phatlc = Iso, Leu, or VaI, Xaa _amphoteπc = His, Arg, GIu, GIn, Asp, or Asn, Xaa^ _Q = Ala or GIn, Xaa _aromatlc = Phe, Tyr, Trp, or His, X ^aa _H p _hob i _c = Iso, Leu, VaI, Cys, Ala, GIy, Met, Phe, Tyr, Trp, or His, Xaa _po i _ar = Tyr, Trp, His, Lys, Arg, GIu, GIn, Asp, Asn, Ser, or Thr, Xaa _Q/ π = GIn or His, Xaa _sma n = VaI, Cys, Ala, GIy, Asp, Asn, Ser, Thr, or Pro, and Xaa _tiny = Ala, GIy, or Ser.

By a "HIAP2 antisense compound" is meant one that decreases the amount of HIAP2 protein by at least about 5%, more desirable by at least about 10%, 25%, or even 50%, relative to an untreated control. Methods for measuring protein levels are well-known in the art. Preferably, a HIAP2 antisense compound of the invention is capable of enhancing apoptosis and/or decreasing HIAP2 protein levels when present in a cell that normally does not undergo sufficient apoptosis. Preferably the increase is by at least 10%, relative to a control, more preferably 25%, and most preferably 1-fold or more. Preferably a HIAP2 antisense compound of the invention includes from about 8 to 30 nucleobases. A HIAP2 antisense compound of the invention may also contain, for example, an additional 20, 40, 60, 85, 120, or more consecutive nucleobases that are complementary to a HIAP2 polynucleotide. The HIAP2 antisense compound (or a portion thereof) may contain a modified backbone. Phosphorothioate, phosphorodithioate, and other modified backbones are known in the art. The HIAP2 antisense compound may also contain one or more non-natural linkages.

By "HIAP2 siRNA" is meant a double stranded RNA comprising a region complementary to a HIAP2 mRNA. Optimally, a HIAP2 siRNA is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length and, optionally, has a two base overhang at one of its 3' end. HIAP2 siRNAs can be introduced to an individual cell, tissue, organ, or to a whole animals. Most preferably, a HIAP2 siRNA is between 21 and 29 nucleotides in length. HIAP2 siRNAs may be introduced systemically via the bloodstream. Such HIAP2 siRNAs are used to downregulate HIAP2 mRNA levels or promoter activity. Desirably, the HIAP2 siRNA is capable of decreasing the expression or biological activity of a nucleic acid or amino acid sequence. In one embodiment, the decrease in expression or biological activity is at least 10%, relative to a control, more desirably 25%, and most desirably 50%, 60%, 70%, 80%, 90%, or more. The HIAP2 siRNA may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.

By "positioned for expression" is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule). By "promoter" is meant a polynucleotide sufficient to direct transcription.

3' regions of the native gene. For example, any polynucleotide region upstream of a gene or a region of an mRNA that is sufficient to direct gene transcription.

"Protein" or "polypeptide" or "peptide" means any chain of more than two natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein.

As used herein, a natural amino acid is a natural α-amino acid having the L- configuration, such as those normally occurring in natural proteins. Unnatural amino acid refers to an amino acid, which normally does not occur in proteins, e.g., an epimer of a natural α-amino acid having the L configuration, that is to say an amino acid having the unnatural D-configuration; or a (D,L)-isomeric mixture thereof; or a homologue of such an amino acid, for example, a β-amino acid, an α, α -disubstituted amino acid, or an α-amino acid wherein the amino acid side chain has been shortened by one or two methylene groups or lengthened to up to 10 carbon atoms, such as an α-amino alkanoic acid with between 5 and 10 carbon atoms in a linear chain, an unsubstituted or substituted aromatic (α-aryl or α-aryl lower alkyl), for example, a substituted phenylalanine or phenylglycine.

The present invention also provides derivatives of the peptides of the invention. Such derivatives may be linear or circular, and include peptides having unnatural amino acids. Derivatives of the invention also include molecules wherein a peptide of the invention is non-covalently or preferably covalently modified by substitution, chemical, enzymatic or other appropriate means with another atom or moiety including another peptide or protein. The moiety may be "foreign" to a peptide of the invention as defined above in that it is an unnatural amino acid, or in that one or more natural amino acids are replaced with another natural or unnatural

amino acid. Conjugates comprising a peptide or derivative of the invention covalently attached to another peptide or protein are also encompassed herein. Attachment of another moiety may involve a linker or spacer, e.g., an amino acid or peptidic linker. Derivatives of the invention also included peptides wherein one, some, or all potentially reactive groups, e.g., amino, carboxy, sulfhydryl, or hydroxyl groups are in a protected form.

The atom or moiety derivatizing a peptide of the invention may serve analytical purposes, e.g., facilitate detection of the peptide of the invention, favor preparation or purification of the peptide, or improve a property of the peptide that is relevant for the purposes of the present invention. Such properties include, cellular uptake, binding to an IAP, or suitability for in vivo administration, particularly solubility or stability against enzymatic degradation. Derivatives of the invention include a covalent or aggregative conjugate of a peptide of the invention with another chemical moiety, the derivative displaying essentially the same activity as the underivatized peptide of the invention, and a "peptidomimetic small molecule" which is modeled to resemble the three-dimensional structure of any of the amino acids of the invention. Examples of such mimetics are retro-inverso peptides (Chorev et al., Ace. Chem. Res. 26: 266-273, 1993). The designing of mimetics to a known pharmaceutically active compound is a known approach to the design of drugs based on a "lead" compound. This may be desirable, e.g., where the "original" active compound is difficult or expensive to synthesize, or where it is unsuitable for a particular mode of administration, e.g., peptides are considered unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Additional examples of derivatives within the above general definitions include the following:

(I) Cyclic peptides or derivatives including compounds with a disulfide bridge, a thioether bridge, or a lactam. Typically, cyclic derivatives containing a disulphide bond will contain two cysteines, which may be L-cysteine or D-cysteine. Advantageously, the N-terminal amino acid and the C-terminal amino acids are

both cysteines. In such derivatives, as an alternative to cysteine, penicillamine (β,β-dimethyl-cysteine) can be used. Peptides containing thioether bridges are obtainable, e.g., from starting compounds having a free cysteine residue at one end and a bromo-containing building block at the other end (e.g., bromo-acetic acid). Cyclization can be carried out on solid phase by a selective deprotection of the side chain of cysteine. A cyclic lactam may be formed, e.g., between the γ-carboxy group of glutamic acid and the ε-amino group of lysine. As an alternative to glutamic acid, it is possible to use aspartic acid. As an alternative to lysine, ornithine or diaminobutyric acid may be employed. Also, it is possible to make a lactam between the side chain of aspartic acid or glutamic acid at the C-terminus and the α-amino group of the N-terminal amino acid. This approach is extendable to β-amino acids (e.g., β-alanine). Alternatively, glutamine residues at the N- terminus or C-terminus can be tethered with an alkenedyl chain between the side chain nitrogen atoms (Phelan et al., J. Amer. Chem. Soc. 119:455-460, 1997). (II) Peptides of the invention, which are modified by substitution. In one example, one or more, preferably one or two, amino acids are replaced with another natural or unnatural amino acid, e.g., with the respective D-analog, or a mimetic. For example, in a peptide containing Phe or Tyr, Phe or Tyr may be replaced with another building block, e.g., another proteinogenic amino acid, or a structurally related analogue. Particular modifications are such that the conformation in the peptide is maintained. For example, an amino acid may be replaced by a α, α - disubstituted amino acid residue (e.g., α-aminoisobutyric acid, 1-amino- cyclopropane-1-carboxylic acid, 1-amino-cyclopentane-l-carboxylic acid, 1 - amino-cyclohexane- 1 -carboxylic acid, 4-amino piperidine-4-carboxylic acid, and 1-amino-cycloheptane-l -carboxylic acid).

(Ill) Peptides of the invention detectably labeled with an enzyme, a fluorescent marker, a chemiluminescent marker, a metal chelate, paramagnetic particles, biotin, or the like. In such derivatives, the peptide of the invention is bound to the conjugation partner directly or by way of a spacer or linker group, e.g., a (peptidic) hydrophilic spacer. Advantageously, the peptide is attached at the

N- or C-terminal amino acid. For example, biotin may be attached to the N- terminus of a peptide of the invention via a serine residue or the tetramer Ser-Gly- Ser-Gly.

(IV) Peptides of the invention carrying one or more protecting groups at a potentially reactive side group, such as amino-protecting group, e.g., acetyl, or a carboxy-protecting group. For example, the C-terminal carboxy group of a compound of the invention may be present in form of a carboxamide function. Suitable protecting groups are commonly known in the art. Such groups may be introduced, for example, to enhance the stability of the compound against proteolytic degradation.

By a "derivative" of a peptide of the invention is also meant a compound that contains modifications of the peptides or additional chemical moieties not normally a part of the peptide. Modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Methods of derivatizing are described below.

Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.

Histidyl residues are generally derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing

α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylissurea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK _a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'~N~C~N~R') such as l-cyclohexyl-3-(2- morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3 (4 azonia 4,4-dimethylpentyl) carbodiimide. Aspartyl and glutamyl residues can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. Peptides of the invention or derivatives thereof may be fused or attached to another protein or peptide, e.g., a protein or peptide serving as internalization vector, such as another peptide facilitating cellular uptake, e.g., a "penetratin." An exemplary penetratin-containing derivative according to the invention is, e.g., a peptide comprising the sixteen amino acid sequence from the homeodomain of the Antennapedia protein (Derossi et al., J. Biol. Chem. 269:10444-10450, 1994), particularly a peptide having the amino acid sequence: Met-Pro-Arg-Phe-Met-Asp- Tyr-Trp-Glu-Gly-Leu-Asn-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn- Glu-Arg-Arg- Met-Lys-Trp-Lys-Lys (SEQ ID NO: 4), or including a peptide sequence disclosed by Lin et al. (J. Biol. Chem. 270:14255-14258, 1995).

Polypeptides or derivatives thereof may be fused or attached to another protein or peptide, e.g., as a glutathione-S-transferase (GST) fusion polypeptide. Other commonly employed fusion polypeptides include, but are not limited to, maltose-binding protein, Staphylococcus aureus protein A, polyhistidine, and cellulose-binding protein.

By a "peptidomimetic small molecule" of a peptide is meant a small molecule that exhibits substantially the same BIR-binding properties as the peptide itself.

By "candidate compound" is meant a chemical, be it naturally- occurring or artificially-derived, that is assayed for its ability to modulate a polypeptide-peptide or protein-protein interaction, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof. By "assaying" is meant analyzing the effect of a treatment, be it chemical or physical, administered to whole animals or cells derived there from. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting altered protein function, protein stability, altered protein-protein interactions, altered protein-peptide interactions, altered protein biological activity. The means for analyzing may include, for example, enzymatic assays, binding assays, irnmunoprecipitation, phosphorylation assays, and methods known to those skilled in the art for detecting nucleic acids and polypeptides.

By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 85%, but preferably 90%, more preferably 95%, most preferably 97%, or even 99% identity to a reference amino acid or nucleic acid sequence.

Sequence identity is typically measured using a sequence analysis program (e.g., BLAST 2; Tatusova et al., FEMS Microbiol Lett. 174:247-250, 1999) with the default parameters specified therein. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine,

isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine and tyrosine.

By "modulating" is meant conferring a change, either by decrease or increase, in IAP biological activity that is naturally present within a particular cell or sample. Preferably, the change in response is at least 5%, more preferably, the change in response is 20% and most preferably, the change in response level is a change of more than 50% relative to the levels observed in naturally occurring IAP biological activity.

By "substantially pure polypeptide" is meant a polypeptide or peptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the polypeptide is an IAP polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure IAP polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding an IAP polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.

By "substantially pure DNA" is meant DNA that is free of the genes that, in the naturally-occurring genome of an organism from which the DNA of the invention might be derived, flank the gene. The term therefore includes, for

example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By "detectably-labeled" is meant any means for marking and identifying the presence of a molecule, e.g., a BIR domain-interacting peptide, a BIR domain polypeptide, a nucleic acid encoding the same, or a peptidomimetic small molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as ³² P or ³⁵ S) and nonradioactive labeling (e.g., chemiluminescent labeling or fluorescein labeling).

If an analysis involves identifying more than one distinct molecule, the molecules can be differentially labeled using markers, which can distinguish the presence of multiply distinct molecules. For example, a BIR domain-interacting peptide can be labeled with fluorescein and a BIR domain polypeptide can be labeled with Texas Red. The presence of the BIR domain- interacting peptide can be monitored simultaneously with the presence of the BIR domain.

The invention features methods and compositions for inducing apoptosis in a cell. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. IA- IF depict immunoblots that show ER stressors induce early processing of caspase-2, downregulate XIAP and HIAPl and upregulate HIAP2. HeLa cells were exposed to BFA and tunicamycin for 24 h (Figs IA and IB) or various other times (Figs. 1C- IF) at the indicated dosages, and cells were then harvested and proteins extracted. Equal proteins (10 or 30 μg) were separated by SDS-PAGE and transferred to nitrocellulose for western immunoblotting with the indicated antibodies. Bound antibodies were detected by the Odyssey® Infrared

Imaging system. Results are representative of those obtained in at least three different experiments. BiP is a marker for ER stress and cleavage of PARP is indicative of activation of the apoptotic pathway.

Fig. 2A depicts an immunoblot showing detection of caspase-2 in the ER and processing of microsomal caspase-2 following ER stress. HeLa cells were exposed to either 400 ng/mL BFA or 20 μg/mL tunicamycin for 24 h. Cells were harvested and mitochondria, cytosol and microsomes were isolated by subcellular fractionation. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose for western immunoblotting with the indicated antibodies. mtHSP70 and SREBP-I are proteins localized to the mitochondria and the ER, respectively, and thus serve as markers of organelle specific preparations.

Figs. 2B-2D are confocal microscopy images of immunofluorescence of endogenous caspase-2 (Fig. 2B), ER-Tracker (Fig. 2C), and a merge of the two (Fig. 2D) in HeLa cells. Figs. 3A-3C show an immunoblot that demonstrates caspase-2 inhibition and siRNA-mediated reduction of caspase-2 expression decrease ER stress-induced apoptosis. In Fig. 3 A, HeLa cells were transfected with the indicated siRNAs, and reduction of caspase-2 level by the respective siRNA was assessed. In Fig. 3B, siRNA-transfected HeLa cells were exposed to 400 ng/mL BFA or 20 μg/mL tunicamycin for 24h. In Fig. 3C, HeLa cells were exposed to 400 ng/mL BFA or 20 μg/mL tunicamycin in the presence or absence of VDVAD-fmk or FA-fmk for 24 h. The expression of proteins was determined by immunoblotting with the indicated antibodies.

Fig. 4A depicts an immunoblot showing that the BIR2 domain of HIAP2 binds caspase-2. GST-tagged BIR domains of XIAP, HIAPl , HIAP2 and GST without a BIR domain were prepared by purification on glutathione- sepharose beads. Each bead-bound GST fusion protein was mixed with recombinant active caspase-2. Resulting complexes were separated by SDS-polyacrylamide gel electrophoresis and the presence of the 19 kDa fragment of caspase-2 confirmed by western immunoblotting.

Figs. 4B-4D are graphs showing that the BIR2 domain of HIAP2 inhibits caspase-2. Caspase-2 activity was assessed in the presence or absence of GST- tagged BIR domains of XIAP (Fig. 4B), HIAPl (Fig. 4C), HIAP2 (Fig. 4D) or GST without a BIR domain (Figs. 4B-4D). VDVAD-pNA hydrolysis by caspase-2 was detected by spectrophotometry. The addition of GST-HIAP2-BIR2 inhibits caspase-2 activity (Fig. 4D). Results are representative of at least three separate experiments.

Figs. 5A-5D are graphs showing adeno-hiap2 protects cells from ER stress- induced cell death. HeLa cells (Figs. 5A and 5B) and SF295 cells (Figs. 5C and 5D) were infected with adeno-lacZ or -hiap2 at a MOI of 5. After 24 h of infection the cells were exposed to BFA (Figs. 5A and 5C) or tunicamycin (Figs. 5B and 5D), and viability was determined 24 h or 48 h later by WST-I assay. Numbers represent the average +/- SEM of three (SF295) or four (HeLa) independent experiments, each performed in triplicate. P < 0.05 (#); P < 0.01 (*) significantly different from uninfected and adeno-lacZ infected, ANOVA with post-hoc Tukey's HSD.

Figs. 6A-6B depict immunoblots showing adeno-hiap2 prevents PARP cleavage without affecting the expression of BiP. HeLa cells were infected with adenoviruses at a MOI of 5. After 24 h the cells were exposed to BFA (Fig. 6A) or tunicamycin (Fig. 6B) at the indicated concentrations for an additional 24 h. The expression of proteins was determined by immunoblotting with the indicated antibodies.

Fig. 6C is a graph showing adeno-hiap2 inhibits caspase-2 activity. HeLa cells were infected with adeno-lacZ or adeno-hiap2 (MOI = 5) for 24 h and then exposed to 20 μg/mL tunicamycin for 18 h. Cytosolic extracts were used for measuring caspase-2 activity. VDVAD-pNA hydrolysis by caspase-2 was detected by spectrophotometry and the percentage activity relative to lysates from non- infected, tunicamycin-treated cells (100%) was plotted. Results are the mean ± SEM (n = three separate experiments). P < 0.001 (*) significantly different from uninfected and adeno-lacZ infected, ANOVA with post-hoc Tukey's HSD.

Fig. 7 A shows an immunoblot that demonstrates that adeno-hiap2as reduces HIAP2 protein levels. HeLa cells were infected with adenoviruses for LacZ (adv- LacZ) or antisense HIAP2 (adv-hiap2as) at a MOI of 5. Cells were harvested at various times and the expression of proteins was determined by immunoblotting. Protein levels of β-actin served as a loading control.

Figs. 7B-7E are graphs showing that adeno-hiap2as sensitizes cell death triggered by ER stress. HeLa cells (Figs. 7B and 7C) and SF295 cells (Figs. 7D and 7E) were infected with adenoviruses for 48 h at a MOI of 5 prior to the induction of apoptosis triggered by BFA (Figs. 7B and 7D) or tunicamycin (Figs. 7C and 7E), and viability was determined 24 h or 48 h later by WST- 1 assay. P < 0.001 (*) significantly different from uninfected and adeno-lacZ infected, ANOVA with post-hoc Tukey's HSD.

DETAILED DESCRIPTION OF THE INVENTION Recent studies have shed light on the importance of the ER as a modulator of mitochondrion-mediated apoptosis (Scorrano et al., Science 300: 135-139, 2003), as well as an organelle-specific, unique pathway for caspase activation and apoptosis (Nakagawa et al., J. Cell Biol. 150:887-894, 2000; Rao et al., J. Biol. Chem. 276:33869-33874, 2001; Morishima et al., J. Biol. Chem. 277: 34287- 34294, 2002; Rao et al., J. Biol. Chem. 277:21836-21842, 2002; Zong et al., J. Cell Biol. 162:59-69, 2003). Here, we delineate an ER stress-induced caspase cascade in humans that is initiated by caspase-2. Given the centrality of IAPs in the regulation of caspase activity, we focused on the potential role of the IAPs in caspase 2-mediated cell death following ER stress. Our results demonstrate that HIAP2-BIR2 directly binds and inhibits casρase-2 and that HIAP2 inhibits ER stress-induced caspase-2 activity, thus providing a novel paradigm for HIAP2 as a specific regulator in the early stages of ER stress-initiated programmed cell death.

We further discovered that ER stress differentially affects the levels of IAPs, downregulating XIAP and HIAPl while upregulating HIAP2. Several competing processes are known to work in concert to dynamically regulate IAP levels and

might be responsible for the observed ER stress-induced changes. In response to ER stress, cap-dependent protein biosynthesis is repressed. Some cellular mRNAs, such as the XIAP- and HIAP2-encoding transcript, contain an internal ribosome entry sequence (IRES) within their 5 '-untranslated regions that allow 5'cap- independent initiation. The HIAP2 IRES element is inducible by ER stress and may partially explain the increased level of HIAP2 protein. In contrast, the XIAP IRES appears to be non-responsive to ER stress. In addition to translational regulation, protein degradation also affects the relative cellular concentration of IAPs following stress. The C-terminal RING domain of XIAP, HIAPl and HIAP2 possesses ubiquitin E3 ligase activity that catalyses auto-ubiquitination as well as the ubiquitination of IAP- interacting proteins. IAPs are also subjected to enzymatic processing by caspases and 0mi/Htra2, a mitochondrial serine protease that also localizes to the ER. In SHSY5Y neuroblastoma cells, Omi/Htra2 protein levels are increased in response to tunicamycin. Alterations in the levels of IAP proteins following ER stress may therefore be explained by a combination of proteolytic processing, auto-ubiquitination and differentially affected IRES-mediated translation.

We propose here that due to its spatial proximity to the site of the initial stress event, and its processing at an early time point, caspase-2 is an apical caspase in the amplification of the caspase cascade following ER stress, analogous to caspase-8 in death receptor-mediated apoptosis. Although ER-specific caspase- 12 has also been implicated as an important apoptotic mediator in response to insults to the ER, human caspase- 12 is presumably functionally inactive due to a mutation of the active site cysteine, rendering this caspase to be an unlikely component in the signaling pathway initiated by ER stress. Recently, caspase-4 has also been implicated as an ER stress-specific caspase in humans. Our current findings suggest that a caspase 2-initiated ER stress-induced apoptotic pathway might operate concomitantly with a caspase 4-mediated pathway.

Although cleaved caspase-2 that has been assembled into a (pl9/pl2) ₂ dimer is active in solution, proteolytic processing per se might not be absolutely required

for activation. An alternative, processing-independent mode of activation for caspase-2 occurs in a "PIDDosome" complex that contains the adaptor protein RAIDD and the death domain-containing protein PIDD. In response to mitochondria-mediated apoptosis, an analogous caspase 9-containing apoptosome that contains Apaf-1, cytochrome c and dATP is formed. XIAP may inhibit the functions of the apoptosome, but it is not known how the PIDDosome may be negatively regulated. Our results demonstrate the ability of the HIAP2-BIR2 domain and/or the HIAP2 BIR1-BIR2 linker region to directly bind to the 19 kDa fragment of caspase-2, thereby suggesting that HIAP2 is a candidate inhibitor of this caspase-2 containing complex.

Mounting evidence implicates ER stress in the pathogenesis of tissue damage after cerebral ischemia, acute renal failure, alcoholic liver injury, diabetes, lysosomal storage disorders, and neurodegenerative disorders such as Alzheimer's, Huntington's and Parkinson's disease. The present results indicate that caspase-2 occupies an important position in ER stress-induced apoptosis caused by BFA and tunicamycin, which target ER-Golgi transport and protein N-linked glycosylation, respectively. The ability of the HIAP2-BIR2 domain to suppress caspase-2 activity suggests that this interaction might serve as the precursor for the discovery of effective caspase-2 inhibitors, facilitating the design and development of therapeutics against various degenerative disorders.

Screening Assays

The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules, or other drugs) that bind HIAP2 or have a stimulatory or inhibitory effect on HIAP2 expression or activity. Compounds thus identified can be used to modulate the activity of HIAP2 in a therapeutic protocol.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art,

including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: De Witt et al. (1993) Proc. Natl. Acad. Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. MoI. Biol. 222:301-310; Ladner supra.). In one embodiment, an assay is a cell-based assay in which a cell which expresses HIAP2 or biologically active portion thereof is contacted with a candidate compound, and the ability of the test compound to modulate HIAP2 biological activity is determined. Determining the ability of the candidate compound to modulate HIAP2 biological activity can be accomplished by monitoring, for example, HIAP2 binding to caspase-2 or caspase-12. The ability of candidate compound to modulate HIAP2 binding to a caspase can be accomplished,

for example, by coupling HIAP2 (or a caspase-interacting portion thereof) or a caspase (or a HIAP2 BIR2- or HIAP2 BIR1-BIR2 linker region-interacting portion thereof) with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 52906, 33408, or 12189 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 52906, 33408, or 12189 could be coupled with a radioisotope, enzymatic, colorimetric, or fluorescent label to monitor the ability of a test compound to modulate HIAP2 binding to a caspase.

In yet another embodiment, a cell-free assay is provided in which HIAP2 or a biologically active portion thereof is contacted with a candidate compound and the ability of the test compound to bind HIAP2 or the biologically active portion thereof is evaluated. Preferred biologically active portions of HIAP2 include the BIR2 domain and/or the HIAP2 BIR1-BIR2 linker region.

Soluble and/or membrane-bound forms of isolated proteins can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton X-100, Triton X-114, Thesit, CHAPS, CHAPSO, and N- dodecyl=N,N-dimethyl-3-ammonio-l -propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected. The interaction between two molecules can be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the donor protein molecule may simply utilize the natural fluorescent

energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the acceptor molecule label may be differentiated from that of the donor. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the acceptor molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the ability of HIAP2 to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338- 2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). BIA detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

It may be desirable to immobilize one of the components being used in the interaction assay to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a candidate compound to HIAP2 or a polypeptide containing a HIAP2 BIR2, HIAP2 BIR1-BIR2 linker region, or HIAP2 RING domain, or interaction of HIAP2 (or a polypeptide containing a HIAP2 BIR2, HIAP2 BIR1-BIR2 linker region, or HIAP2 RING domain) with a caspase (or a polypeptide containing a HIAP2 BIR2- or HIAP2 BIR1-BIR2 linker region-interacting portion thereof) in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/HIAP2

fusion proteins or glutathione-S-transferase/caspase-2 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the candidate compound or the candidate compound and either the non-adsorbed HIAP2 or caspase (or fragment thereof), and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.

Other techniques for immobilizing a compound on matrices include using conjugation of biotin and streptavidin. Biotinylated compounds can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non- immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with HIAP2 or the interacting caspase (e.g., caspase-2, caspase-3, caspase-4, caspase-7, or caspase- 12) but which do not interfere with binding of HIAP2 to the

caspase. Such antibodies can be derivatized to the wells of the plate. Methods for detecting such complexes, in addition to those described above for the GST- immobilized complexes, include immunodetection of complexes using antibodies reactive with the HIAP2 or the caspase. Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion- exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J MoI Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In a preferred embodiment, the assay includes contacting HIAP2 or a biologically active portion thereof with caspase-2 to form an assay mixture, contacting the assay mixture with a candidate compound, and determining the ability of the candidate compound to interact with HIAP2, wherein determining the ability of the candidate compound to interact with HIAP2 includes determining the ability of the test compound to preferentially bind to HIAP2 or a biologically active portion thereof.

HIAP2 can interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity of HIAP2. Such compounds can include, but are not limited to, molecules

such as antibodies, peptides, and small molecules. Particularly useful portions of HIAP2 are the BIR domain, the BIR1-BIR2 linker region, and the RING domain. To identify compounds that interfere with the interaction between a HIAP2 and its cellular or extracellular binding partner(s), a reaction mixture containing a HIAP2 (or a polypeptide containing a HIAP2 BIR2, HIAP2 BIR1-BIR2 linker region, or HIAP2 RING domain) and the binding partner is prepared under conditions and for a time sufficient to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of a candidate compound. The candidate compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the candidate compound or with a placebo. The formation of any complexes between the HIAP2 or HIAP2 fragment and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the candidate compound, indicates that the compound interferes with the interaction of the HIAP2 (or a polypeptide containing HIAP2 BIR2, HIAP2 BIR1-BIR2 linker region, or HIAP2 RING domain) and the binding partner.

In another embodiment, modulators of HIAP2 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of HIAP2 mRNA or protein evaluated relative to the level of expression of HIAP2 mRNA or protein in the absence of the candidate compound. When expression of HIAP2 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of HIAP2 mRNA or protein expression. Alternatively, when expression of HIAP2 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of HIAP2 mRNA or protein expression.

EXAMPLES

The following examples are meant to illustrate the invention, are not intended to be limiting.

Example 1: ER stress induces apoptosis, downregulates XIAP and HIAPl proteins, but upregulates HIAP2

The ability of BFA and tunicamycin to trigger ER stress by inhibiting ER- Golgi transport and by blocking the formation of protein N-glycosidic linkages, respectively, is well documented (Kaufman, Genes Dev 13: 1211-1233, 1999; Patil et al, Curr. Opin. Cell Biol. 13:349-355, 2001). The induction of the ER chaperone BiP is an established marker for the presence of ER stress (Imaizumi et al., Biochim. Biophys. Acta 1536: 85-96, 2001 ; Gulow et al., J. Cell Sci. 115:2443- 2452, 2002). To confirm induction of ER stress, protein levels of BiP were assessed by immunoblotting cell extracts with specific antibodies. As expected, BiP protein levels were elevated in HeLa cells in response to BFA and tunicamycin, thereby confirming the induction of ER stress in these cells (Figs. IA- ID). To determine whether ER stress resulted in the activation of apoptosis, we analysed the cleavage of the caspase substrate PARP. Treatment with ER stressors resulted in the processing of full-length PARP (116 kDa) to its cleaved 89 kDa form (Figs. IA- ID), indicating that apoptosis was induced. We next examined the effect of ER stress on IAP protein levels. Protein levels of HIAP2 were upregulated in response to the ER stressors, with the exception of prolonged or high concentration exposure to tunicamycin. In contrast, protein levels of both XIAP and HIAPl decreased in a time- and dose-dependent manner (Figs. 1 A-ID). The processing of PARP indicated that ER stress activates caspase- dependent apoptotic programs. We therefore assessed the temporal onset of caspase cascade initiated by ER stress. Caspase-2 has previously been implicated as an initiator caspase in both the intrinsic and extrinsic pathways. We therefore investigated the possibility that caspase-2 may be an early mediator of ER stress- induced apoptosis. Processing of caspase-2, as well as the initiator caspase-9, was

seen at 6 h of ER stress, followed by the activation of the executioner caspase-7 at 12 h (Figs. IE and IF). Earlier studies showed that under apoptotic conditions, including exposure to UV, TNF, staurosporine or Fas ligands, caspase-2 activation occurs subsequent to caspase-3. Following ER stress, however, we observed that the cleavage of caspase-2 occurred prior to the activation of caspase-3 (Fig. IE and IF), indicating that at early time points, caspase-2 activation is independent of caspase-3. In fact, caspase-2 cleavage preceded that of all of the other tested caspases, implicating caspase-2 as the apical caspase in ER-pathway specific apoptosis.

Example 2: Identification of ER-associated caspase-2

In rodent cells, microsome-associated caspase- 12 is involved in the apoptotic cascade elicited by ER stressors (Nakagawa et al., J. Cell Biol. 150:887- 894, 2000; Nakagawa et al., Nature 403:98-103, 2000; Rao et al., J. Biol. Chem. 276:33869-33874, 2001; Yoneda et al. J. Biol. Chem. 276: 13935-13940, 2001). However, the human homologue of rodent caspase- 12 contains a deleterious mutation, suggesting that a different apical caspase is involved in humans (Fischer et al., Biochem. Biophys. Res. Commun. 293: 722-726, 2002). The above results suggested that caspase-2 may be the apical caspase that mediates ER stress-induced apoptosis, and thus we further examined the intracellular localization of caspase-2. A subpopulation of caspase-2 is found in ER-enriched microsomal fractions (Paroni et al., J. Biol. Chem. 277: 15147-15161, 2002; Fig. 2A). Subcellular fractionation revealed that treatment of cells with BFA or tunicamycin for 24 h induced processing of pro-caspase-2 in the mitochondrial, cytosolic and ER-enriched microsomal fractions, whereas cleaved caspase-2 was detected only in the cytosol (Fig. 2A). In addition, we observed the translocation of full-length caspase-7 to the ER, as well as a salient accumulation of activated caspase-7 in the microsomal fraction (Fig. 2A). Consistent with previous results (Figs. IE and IF), cleaved caspase-3 and -9 were also detected in the cytosol after 24 h of ER stress (Fig. 2A).

To verify the in situ location of caspase-2, we performed immunofluorescence confocal microscopy. Antibodies specific to caspase-2 stained the cytoplasm, the nucleus and the perinuclear region of HeLa cells (Fig. 2B). The existence of caspase-2 in the ER of these cells was confirmed by co- staining with the ER-Tracker Dapoxyl dye (Figs. 2C and 2D).

Example 3: Silencing of caspase-2 decreases ER stress-induced apoptosis

To determine whether caspase-2 is required for ER stress-induced cell death, we used small interfering RNA (siRNA) to silence the expression of caspase-2 prior to the induction of ER stress. The specificity of the casp2-siRNA sequence used in these experiments was characterized previously (Lassus et al., Science 297:1352-1354, 2002; Lin et al., J. Biol. Chem. 279:40755-40761, 2004; Wagner et al. J. Biol. Chem. 279:35047-35052, 2004). Protein expression level of caspase-2 was substantially decreased after 60 h of transfection with casp2-siRNA, but it was not affected by transfection with the non-silencing siRNA, as compared with non- transfected cells (Fig. 3A). Silencing of caspase-2 by siRNA reduced the amount of PARP cleavage induced by BFA and tunicamycin without affecting increased protein expression of BiP, indicating that although ER stress persisted, apoptosis was inhibited (Fig. 3B). To confirm that caspase-2 is involved in apoptosis caused by ER stress, we next exposed HeLa cells to ER stressors in the presence of either the caspase-2 inhibitor VDVAD-fmk or as a control, the cathepsin B inhibitor FA- fmk. ER stress-induced PARP cleavage was reduced by VDVAD-fmk, but BiP induction was not affected by the caspase-2 inhibitor (Fig. 3C). These results demonstrate that decreased expression or activity of caspase-2 renders cells more resistant to ER stress-induced apoptosis.

Example 4: Inhibition of caspase-2 by the HIAP2-BIR2 domain

Previous studies have established that the IAP proteins are capable of binding to and inhibiting caspase-3, -7 and -9; however, it is still unknown whether IAPs also inhibit caspase-2. We therefore examined the ability of IAP proteins to

interact with caspase-2 by assessing the ability of individual IAP-BIR domains to interact with recombinant active caspase-2. Immobilized GST-IAP-BIR domains on glutathione- sepharose beads were incubated with recombinant caspase-2, and the resulting complexes were examined by SDS-PAGE and Western blot analysis. Among the different BIR domains of XIAP, HIAPl and HIAP2, only the BIR2 domain of HIAP2 bound to caspase-2 (Fig. 4A). Some residual HIAPl -BIR2 and XIAP-BIRl interaction with caspase-2 was noted, but not considered significant (Fig. 4A). No binding occurred when GST-sepharose lacking an associated IAP- BIR domain was employed, indicating that binding was specific for the IAPs. To further analyse for the functional significance of these interactions, we assayed for proteolytic activity of recombinant caspase-2 in the presence of the IAP-BIR domains. Caspase 2-directed hydrolysis of its substrate VDVAD-pNA was inhibited by the HIAP2-BIR2 domain but not by any other IAP-BIR domains tested (Fig. 4B-D). GST-XIAP-BIR2 and GST-XIAP-BIR3 inhibit caspase-3 and caspase-9 activity, respectively, as expected, indicating that these recombinant proteins were functionally active. These results highlight a novel relationship between caspase-2 and HIAP2.

Example 5: HIAP2 overexpression rescues cells from ER stress-induced apoptosis

The ability of HIAP2 to inhibit caspase-2 and the early processing of caspase-2 following ER stress lead us next to determine whether HIAP2 over- expression could protect cells from ER stress-induced apoptosis. HeLa cells were plated in 96- well plates and infected with adeno-lacZ or adeno-hiap2. After 24 h, triplicate samples were exposed to BFA or tunicamycin for an additional 24 h and cell viability determined using WST-I reagent. Adeno-hiap2 conferred a significant degree of protection across a wide range of BFA and tunicamycin doses tested as compared with uninfected and adeno-lacZ infected cells (Figs. 5 A and 5B). Similar protection was seen with SF295 glioblastoma cells (Figs. 5C and 5D).

We next assessed the possibility that over-expression of HIAP2 may suppress apoptosis by relieving ER stress. Infection with adeno-hiap2 failed to affect ER stressor-mediated induction of BiP (Fig. 6A and B), confirming that HIAP2 inhibits cell death through a mechanism that is independent of the alleviation of ER stress per se.

To determine if IAP-mediated protection from ER stress involves caspase inhibition, we analysed for the presence of cleaved PARP in lysates from cells infected with adenoviruses. Consistent with the results of the WST-I cell viability assays, processing of PARP was inhibited by adeno-hiap2, but not by adeno-lacZ (Figs. 6A and 6B). Infection with adeno-hiap2 did not alter endogenous expression levels of the other IAPs examined (Figs. 6A and 6B), suggesting that overexpression of HIAP2 alone accounted for the protection.

To further determine the relationship between HIAP2 and ER stress-induced activation of caspase-2, we investigated the effects of HIAP2 overexpression on tunicamycin- induced caspase-2 activity. After 18 h of drug exposure, extracts from cells were analysed for caspase-2-like activity by measuring the rate of VDVAD- pNA hydrolysis. Infection with adeno-hiap2 resulted in a 55% suppression of the caspase-2-like activity relative to uninfected control and adeno-lacZ infection (Fig. 6C).

Example 6: Blocking endogenous HIAP2 expression enhances ER stress- induced cell death

The contribution of endogenous HIAP2 towards the suppression of ER stress-induced killing of cancer cells was investigated using antisense adenovirus specific to hiap2 (adeno-hiap2as). The efficacy of adeno-hiap2as in downregulating HIAP2 was confirmed by western immunoblotting (Fig. 7A). After 48 h of infection by adeno-lacZ or adeno-hiap2as (MOI = 5 for each virus), HeLa cells were then exposed to BFA or tunicamycin for 24 h. Analysis of HeLa cell survival by WST- 1 revealed that infection with adeno-hiap2as virus increased sensitivity to ER stress-mediated cell death (Figs. 7B and 7C). SF295 cells were

similarly sensitized to ER stress when endogenous HIAP2 was downregulated (Fig. 7D and E). These results indicated that endogenous HIAP2 plays a fundamental role in maintaining cell viability following ER stress.

The foregoing results were obtained with the following materials and methods

Cell culture and harvesting

HeLa cervical carcinoma cells (ATCC) and SF295 glioblastoma cells (University of California San Francisco Neurosurgery Tissue Bank) were maintained at 37°C and 5% CO ₂ in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) supplemented with 10% heat- inactivated fetal calf serum (Invitrogen), penicillin, and streptomycin (Invitrogen). Trypsinized cells were collected by centrifugation and lysed in 50 mM Tris-HCl, pH 8.0 containing 1% Triton X- 100, 150 mM NaCl, 1 mM NaF, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5 μg/mL pepstatin A and 10 μg/mL each of leupeptin and aprotinin (lysis buffer) and insoluble cell debris pelleted by centrifugation at 12,000 x g for 30 min at 4 ⁰ C. Supernatants were collected and protein content determined by Bio-Rad Protein Assay using bovine serum albumin (BSA) as a standard.

Plasmid DNA constructs pGEX-XIAP-BIRl, -BIR2, -BIR3; HIAPl-BIRl, -BIR2, -BIR3; and HIAP2-BIR1, -BIR2 and -BIR3: Individual BIR domains of XIAP were PCR- amplified and TA-cloned as described. BIR domains of HIAPl and HIAP2 were PCR-amplified and TA cloned using the following primers: HIAPl-BIRl 5'-d- GGATCC ATGAACATAGTAGAAAACAGCATATTC-S' (SEQ ID NO: 5) and 5 '-d-CTCGAGTC AA ACGA ATCTGC AGCT AGG AT AC-3' (SEQ ID NO: 6); HIAP1-BIR2 5 '-d-GG ATCC ATGC AG AGTCT A A ATTCCG- 3' (SEQ ID NO: 7) and 5 '-d-CTCGAGTT AT AT A A ATGGGC ATTTGGG AAAATGTC-3' (SEQ ID NO: 8); HIAP1-BIR3 5 '-d-C AGGATCC ATGG AA AATC AGCTTC AAGAC-3'

(SEQ ID NO: 9) and S'-d-TGCTCGAGTTATGATGTGGATAGCAGCTG-S' (SEQ ID NO: 10); HIAP2-BIR1 5'-d-GGATCCATGCACAAAACTGCCTCCCA AAGAC-3' (SEQ ID NO: 11) and 5 '-d-CTCG AGTC A AT A A AGCT AC AGCT AG GATATAGC-3' (SEQ ID NO: 12); HIAP2-BIR2 5'-d- AGATCTATGCAGAATCT GGTTTCAGCTAG-S' (SEQ ID NO: 13) and 5'-d- CTCGAGTCAAAAAATGGAC AGTTGGGAA-3' (SEQ ID NO: 14); HIAP2- BIR3 5'-d-CAGGATCCATGGAAA ATTCTCTAGAAACTC-3' (SEQ ID NO: 15) and 5 '-d-TGCTCGAGTT ATGAAG TTGAC AAC AGCTG-3' (SEQ ID NO: 16). The XIAP BIR domain-containing fragments (amino acids 1-123, 124-240, and 241-356), HIAPl BIR domain-containing fragments (amino acids 1-96, 97- 235, and 236-349) and HIAP2 BIR domain-containing fragments (amino acids 1- 113, 114-250, 251-363) were subcloned into pGEX-4T3 using the BamHl and Xhol sites present in the primers.

Preparation and transfection of siRNA

Annealed double stranded siRNA corresponding to the following cDNA sequences were purchased from Qiagen: 5'-AA-ACAGCTGTTGTTGAGCGAA- dTdT-3' (nucleotides 94-114; SEQ ID NO: 17) for caspase-2 and 5'-AA- TTCTCCGAACGTGTC ACGT-dTdT-3' (SEQ ID NO: 18) for non-silencing controls. Cells were cultured in 6- well plates with 2 mL of medium and transfected at 50% confluency with a final concentration of 100 nM siRNAs using RNAiFect Transfection Reagent (Qiagen) according to the manufacturer's protocol. Transfected cells were rinsed with the medium after 16 h of incubation and then maintained in culture for additional 20 h before the addition of either BFA (Sigma, 400 ng/mL), tunicamycin (Sigma, 20 μg/mL), or equivalent concentration of vehicle (0.02% ethanol and 0.1% DMSO, respectively), for 24 h before analysis. Efficiency of siRNA was measured by western blot analysis.

Recombinant protein expression

Recombinant glutathione-S-transferase (GST) proteins fused to individual BIR domains of the IAPs were prepared by purification on glutathione-agarose beads (Amersham Biosciences). In some experiments, the GST-fusion proteins were eluted with 50 mM Tris-HCl, pH 8.0 containing 10 mM reduced glutathione, 150 mM NaCl, 5 mM DTT and 10% glycerol. These proteins were quantified by SDS-polyacrylamide gel electrophoresis using BSA as a protein standard prior to performing pull-down analysis.

In vitro pulldown assay

To analyze IAP binding to caspase-2, 2 μM of GST-fusion protein immobilized on glutathione- sepharose beads was incubated in the presence of IU recombinant active caspase-2 that consists of the 19 kDa and 12 kDa subunits (Calbiochem) in binding buffer (50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 10 mM EDTA, 0.1% Triton-X- 100) at 4 ⁰ C for 1 h on a rotating platform. The beads containing bound proteins were washed three times with binding buffer and bound proteins eluted in 62.5 mM Tris-HCl, pH 6.8 containing 2% SDS, 1% β- mercaptoethanol and 5% glycerol (sample buffer) by heating at 100 ⁰ C for 5 min.

Caspase-2 inhibition assay

Recombinant caspase-2 activity was determined using a colorimetric caspase-2 assay kit pursuant to the manufacturer's instructions (Calbiochem). In short, 2 μM of GST-fusion proteins and IU recombinant caspase-2 were incubated at 37 ⁰ C in the presence of reaction buffer (50 mM Tris-HCl, pH 8.0 containing 250 mM NaCl, 10 mM EDTA, 0.1 % Triton X-100, 1 mM DTT) and caspase-2 activity was measured using 200 μM p-nitroaniline (pNA) conjugated VDVAD peptide substrate. VDVAD-pNA hydrolysis was monitored at 3 min intervals for 2 h using a spectrophotometer. No spontaneous hydrolysis of VDVAD-pNA was detected in the absence of caspase-2 throughout the course of the assay. Endogenous caspase- 2 activity was similarly determined, except that 300 μg lysates from tunicamycin-

treated HeLa cells were used and VDVAD-pNA hydrolysis was detected after a 6 h reaction.

Recombinant adenoviral vectors The preparation of adeno-lacZ and adeno-hiap2 has been previously described (Simons et al., J. Neurochem. 72:292-301,1999). The adenoviral vector for antisense HIAP2 (adeno-hiap2as) was prepared as described previously (Li et al., Endocrinology 142:370-380, 2001).

Survival assays

Survival assays for HeLa cells exposed to ER stressors were performed in 96- well dishes plated with 1000 cells per well in complete DMEM. For experiments involving SF295 cells, 2000 cells per well were plated, except for results presented in Fig. 7D, in which 1000 SF295 cells were plated per well. Cells were infected with the recombinant adenoviruses at an MOI of 5 in all cases. For experiments determining the effects of adenovirus-mediated HIAP2 over- expression, after 24 h of infection, the cells were exposed to the following ER stressors: BFA (0.2 ng/niL to 2 μg/mL for HeLa cells, 24 h; 2 μg/mL for SF295 cells, 48 h) and tunicamycin (0.2 μg/mL to 100 μg/mL for HeLa cells, 24 h; 20 μg/mL for SF295 cells, 48 h) or equivalent concentration of vehicle (0.02% ethanol and 0.1% DMSO, respectively). For experiments determining the effects of antisense adeno-HIAP2, at 48 h post-infection, the cells were treated with BFA (400ng/mL for HeLa cells, 24 h; 2 μg/mL for SF295 cells, 24 h) and tunicamycin (5 μg/mL for HeLa cells, 24 h; 5 μg/mL for SF295 cells, 48 h). At the end of the treatment period, cell viability was determined using the WST- 1 reagent according to the manufacturer's instructions (Boehringer-Mannheim).

Gel electrophoresis and western immunoblotting

For immunoblotting, sodium dodecyl sulfate (SDS)-solubilized samples (10 μg to 30 μg) were separated on 10% or 15% polyacrylamide gels and transferred to

nitrocellulose. Following protein transfer, individual proteins were detected by western immunoblotting using one of the following antibodies: anti-β-actin (Sigma); anti-BiP and anti-SREBP-1 (BD Biosciences); anti-caspase-2 (Chemicon International Inc.); anti-caspase-3, anti-caspase -7, anti-caspase -9, anti-cleaved caspase-3, anti-cleaved caspase -7 and anti-cleaved caspase -9, and anti- PARP (New England Biolabs); anti-HIAPl and anti-HIAP2 (R&D Systems); anti- mitochondrial HSP70 (Affinity BioReagents). Polyclonal anti-XIAP antibody was generated by immunizing rabbits with GST-XIAP fusion protein in RIBI adjuvant (Sigma) as described (Li et al., Endocrinology 142:370-380, 2001). Antiserum was cleared of anti-GST-antibody on a GST-Sepharose column. Bound primary antibodies were reacted with secondary antibodies conjugated with Alexa Fluor® 680 (Molecular Probes) and the infrared fluorescence signals were detected using Odyssey® Infrared Imaging System (LI-COR).

Subcellular fractionation

Fractionation was performed as described previously (Reddy et al., J. Biol. Chem. 274:28476-28483, 1999; Rao et al., J. Biol. Chem. 276:33869-33874, 2001) with some modifications. Briefly, control, 400 ng/niL BFA-treated and 20 μg/mL tunicamycin-treated HeLa cells were resuspended and lysed in ice-cold hypotonic extraction buffer (10 mM Tris-HCl, pH 7.4, 50 raM KCl) using a B-type pestle. The lysate was immediately adjusted to 250 mM sucrose, 1 mM MgCl ₂ , 0.5 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 5 μg/mL pepstatin A, 10 μg/mL each of leupeptin and aprotinin and centrifuged at 750 x g for 10 min at 4°C to remove nuclei and cell debris. The supernatant was further centrifuged at 10,000 x g for 30 min at 4 ⁰ C. The pellet representing the mitochondrial fraction was resuspended in the above buffer. The supernatant was re-centrifuged at 100,000 x g for 60 min. The resulting supernatant contained the soluble cytoplasmic fraction, and the pellet, constituting the ER-enriched microsomal fraction, was rinsed and resuspended as above. The quality of the fractionation experiments was controlled by assessing the distribution of mtHSP70 for mitochondria and SREBP- 1 for ER, respectively.

Confocal microscopy

For immunofluorescence confocal microscopy, overnight HeLa cell cultures were treated with 2 μM ER-Tracker Dapoxyl dye (Molecular Probes) at 37 ⁰ C for 1 h. After exposure to the ER-Tracker, cells were fixed in methanol at -2O ⁰ C for 5 min, and then incubated first with anti-caspase-2 antibody followed by Texas Red- conjugated secondary antibody (Molecular Probes). Fluorescence-labeled samples were mounted in ProLong® Antifade (Molecular Probes) and visualized using a Nikon Cl laser scanning confocal microscope equipped with a titanium sapphire laser (Spectra-Physics Lasers and Photonics).

Statistical analysis

Statistical comparisons were made using an ANOVA with post-hoc Tukey's HSD.

Other Embodiments

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the invention. What is claimed is: