Showing papers by "Emad S. Alnemri published in 1996"

In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.

Abstract: Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.

Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

Abstract: The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the pre-cursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Abstract: Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis.

Abstract: Radiosensitive cell lines derived from X-ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA-dependent protein kinase, DNA-PK, suggesting that DNA-PK plays an important role in DNA double-strand break repair. Another enzyme implicated in DNA repair, poly(ADP-ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA-PKcs, the catalytic subunit of DNA-PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA-binding component of the enzyme, remains intact. Degradation of DNA-PKcs was accompanied by loss of DNA-PK activity. One cell line resistant to etoposide-induced apoptosis failed to show degradation of DNA-PKcs. Protease inhibitor data implicated an ICE-like protease in the cleavage of DNA-PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA-PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA-PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA-PKcs is a critical target for proteolysis by an ICE-like protease during apoptosis.

Cooperative action of Hsp70, Hsp90, and DnaJ proteins in protein renaturation.

Abstract: The proteins required for the repair of damaged proteins in the eukaryotic cytoplasm remain largely uncharacterized. The renaturation of thermally denatured firefly luciferase readily occurs in rabbit reticulocyte lysate by an ATP-dependent process. Earlier studies had shown that this chaperoning activity could be reconstituted, in part, using purified preparations of hsp70 and hsp90. We have extended the description of this system by clarifying the importance of hsp70 and hsp90 and have tested for additional factors that enhance renaturation. Using mutant hsp70 proteins, we have shown that hsp70 is required for luciferase renaturation. We have also found that hsp70 and hsp90 preparations purified by common procedures were contaminated with low levels of DnaJ proteins that are essential for the renaturing activity. When hsp70 and hsp90 preparations free of DnaJ proteins are used, the system must be supplemented with a DnaJ protein to obtain renaturation activity. The yeast DnaJ protein, YDJ-1, was found to be very effective for this purpose. Although significant renaturation can occur with only hsp70 and DnaJ proteins, hsp90 also contributes to the renaturation process, both in the complex environment of reticulocyte lysate and in a purified system. However, using highly purified hsp90 and geldanamycin, a specific inhibitor of hsp90 function, we have determined that hsp90 is not an essential component of the renaturation system. The contribution of hsp90 to renaturation is only partially blocked by geldanamycin, suggesting that this protein may influence activity in more than one way. This study indicates that hsp70, hsp90, and DnaJ proteins function cooperatively to renature damaged proteins in the eukaryotic cytoplasm and provides a framework by which additional components can be identified and individual chaperone contributions can be investigated.

Processing of the Nedd2 precursor by ICE‐like proteases and granzyme B

Abstract: Background: The Nedd2/Ich-1 protein belongs to a growing family of mammalian cysteine proteases similar to interleukin-1β converting enzyme (ICE). Because of their similarity to the Caenorhabditiselegans cell death protein CED-3, the ICE-like proteins are thought to play a key role in the execution of apoptosis. The active form of ICE is a tetramer consisting of two heterodimers (p20 + p10)2 derived from the cleavage of the pro-enzyme. Results: In the present communication we show that the p51 Nedd2 precursor (pro-Nedd2) is also cleaved into p20-like (p19) and p10-like (p12) subunits by extracts prepared from cultured cell lines. Extracts from apoptotic NIH-3T3 cells but not normal growing NIH-3T3 cells also contained pro-Nedd2 cleaving activity. The processing of pro-Nedd2 by cell extracts was inhibited by characteristic inhibitors of ICE-like proteases. Additionally we show that pro-Nedd2 (p51) can be processed in vitro by active CPP32 and ICE, and to a lesser extent by Mch2 and Nedd2. Granzyme B, a serine protease required for cytotoxic T lymphocyte (CTL) mediated killing of target cells, also cleaved pro-Nedd2 to p19 + p12 subunits. Conclusions: Our observations suggest that Nedd2 activation requires cleavage by one or more ICE-like proteases that lie upstream in the proteolytic cascade. Cleavage of pro-Nedd2 by granzyme B indicates that Nedd2 may be one of the downstream effectors in the CTL-mediated killing of target cells.

Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing

Abstract: Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

Mch2, AN APOPTOTIC CYSTEINE PROTEASE, AND COMPOSITIONS FOR MAKING AND METHODS OF USING THE SAME

Abstract: A substantially pure protein that is a member of the apoptotic Ced-3/Ice cysteine protease gene family, Mch2α, and an inactive isoform of it, Mch2β, are disclosed. Isolated nucleic acid molecules that encode Mch2α and Mch2β, respectively, are disclosed. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier in combination with the protein or the nucleic acid molecules are disclosed. Fragments of nucleic acid molecules that encode Mch2α and Mch2β having at least 10 nucleotides and oligonucleotide molecule comprising a nucleotide sequence complimentary to a nucleotide sequence of at least 10 nucleotides are disclosed. Recombinant expression vectors that comprise the nucleic acid molecule that encode Mch2α or Mch2β, and host cells that comprise such recombinant vectors are disclosed. Antibodies that bind to an epitope on Mch2α and/or Mch2β are disclosed. Methods of identifying inhibitors, activators and substrates of Mch2α are disclosed. Antisense compounds and methods of using the same are disclosed.

Isoforms of human interleukin-1β converting enzyme and methods of using the same

Abstract: Substantially pure interleukin-1 converting enzyme isoforms are disclosed. Pharmaceutical compositions comprising one or more interleukin-1 converting enzyme isoforms are disclosed. Nucleic acid molecules that encode interleukin-1 converting enzyme isoforms, recombinant expression vectors that comprise a nucleic acid sequence that encodes an interleukin-1 converting enzyme isoform, and host cells that comprise recombinant expression vectors that comprise nucleic acid sequences that encode interleukin-1 converting enzyme isoforms are disclosed. Fragments of nucleic acid molecules with sequences encoding interleukin-1 converting enzyme isoform and oligonucleotide molecules that comprise a nucleotide sequence complimentary to fragment of a nucleotide sequence that encodes an interleukin-1 converting enzyme isoform are disclosed. Antibodies which bind to an epitope on interleukin-1 converting enzyme isoforms are disclosed. Methods of identifying inhibitors of ICE isoforms are disclosed.

SF caspase-1 and compositions for making and methods of using the same

Abstract: A substantially pure protein, Caspase-1, is disclosed. An isolated nucleic acid molecule that comprises a nucleic acid sequence that encodes Caspase-1, is disclosed. An isolated nucleic acid molecule consisting of a nucleic acid sequence that encodes Caspase-1, or a fragment thereof having at least 10 nucleotides is disclosed. Recombinant expression vector comprising a nucleic acid sequence that encodes Caspase-1 and host cells comprising the recombinant expression vector are disclosed. Oligonucleotide molecule comprising a nucleotide sequence complimentary to a nucleic acid sequence that encodes Caspase-1 of at least 5 nucleotides are disclosed. Antibodies that binds to an epitope on Caspase-1 are disclosed. Methods of identifying modulators and substrates of Caspase-1 are disclosed.

MCH4 and MCH5, apoptotic proteases

Abstract: The invention provides an isolated gene encoding Mch4 or an isolated gene encoding Mch5 as well as functional fragments thereof. Also provided are isolated nucleic acid sequences encoding Mch4 or Mch5 or functional fragment thereof. The gene or nucleic acid sequences can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch4 or Mch5 nucleotide sequences. Isolated Mch4 or Mch5 polypeptides or functional fragments thereof are also provided.

Nucleotides encoding immunophilin FKBP46 and fragments thereof

Abstract: A substantially pure protein, immunophilin FKBP46, is disclosed. An isolated nucleic acid molecule that comprises a nucleic acid sequence that encodes immunophillin FKBP46, is disclosed. An isolated nucleic acid molecule consisting of a nucleic acid sequence that encodes immunophillin FKBP46, or a fragment thereof having at least 10 nucleotides is disclosed. Recombinant expression vector comprising a nucleic acid sequence that encodes immunophillin FKBP46 and host cells comprising the recombinant expression vector are disclosed. Oligonucleotide molecule comprising a nucleotide sequence complimentary to a nucleic acid sequence that encodes immunophillin FKBP46 of at least 5 nucleotides are disclosed. Antibodies that binds to an epitope on FKBP46 are disclosed. Methods of identifying immunosuppressive drugs comprising the steps of contacting FKBP46 or a homologous protein derived from yeast with a test compound and determining whether the protein binds to the test compound are disclosed.