Showing papers by "Emad S. Alnemri published in 2002"

The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity.

Abstract: Proteome analysis of supernatant of isolated mitochondria exposed to recombinant tBid, a proapoptotic Bcl-2 member, revealed the presence of the serine protease Omi, also called HtrA2. This release was prevented in mitochondria derived from Bcl-2-transgenic mice. Release of Omi under apoptotic conditions was confirmed in vivo in livers from mice injected with agonistic anti-Fas antibodies and was prevented in livers from Bcl-2 transgenic mice. Omi release also occurs in apoptotic dying but not in necrotic dying fibrosarcoma L929 cells, treated with anti-Fas antibodies and TNF, respectively. The amino acid sequence reveals the presence of an XIAP interaction motif at the N-terminus of mature Omi. We demonstrate an interaction between endogeneous Omi and recombinant XIAP. Furthermore we show that endogenous Omi is involved in enhanced activation of caspases in cytosolic extracts.

Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi

Abstract: HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.

Caspase-3 expression by cerebellar granule neurons is regulated by calcium and cyclic AMP

Abstract: Caspase-3 enzyme activity is induced, and cell death follows, when cerebellar granule neurons (CGNs) from 8-day-old rats are transferred from an extracellular concentration of 25 mM K + (25 mM [K + ] e ) to 5 mM [K + ] e . Death of these neurons is diminished by an inhibitor of caspase-3 but not by an inhibitor of caspase-1. Actinomycin D and cycloheximide inhibit induction of caspase-3 and prevent death. Experiments in which CGN intracellular Ca 2+ concentration ([Ca 2+ ] i ) was manipulated by either changing [K + ] e or adding a voltage-gated Ca 2+ channel antagonist or a Ca 2+ ionophore to the medium showed that caspase-3 mRNA rises 2.5-fold when [Ca 2+ ] i is diminished from 300 to 150 nM, with a corresponding rise in peak caspase enzyme activity. Whereas the caspase-3 mRNA level does not rise further with a still greater diminution in [Ca 2+ ] i peak caspase enzyme activity continues to increase, reaching sevenfold induction when [Ca 2+ ] i is reduced to 55 nM. In CGNs in which [Ca 2+ ] i is set at 55 nM by incubation in 5 mM [K + ] e , treatment with forskolin or dibutyryl 3',5'-cyclic adenosine-5'-monophosphate delays caspase-3 induction and diminishes death but does not alter [Ca 2+ ] i . We conclude that, in immature CGNs, both caspase-3 transcription and the subsequent processing of caspase-3 are induced by a fall in [Ca 2+ ] i . Elevating cyclic AMP content delays caspase-3 induction by a mechanism that does not require an increase in [Ca 2+ ] i .

Expression and transcriptional regulation of caspase-14 in simple and complex epithelia.

Abstract: Caspase-14 is a recent addition to the caspase family of aspartate proteases involved in apoptotic processes. Human caspase-14 appears to be only weakly processed during apoptosis, and it does not cleave classical caspase substrates. Post partum, caspase-14 is prominently expressed by human keratinocytes and reportedly participates in terminal differentiation of complex epithelia. Here we provide evidence challenging the view that caspase-14 expression or processing is linked exclusively to terminal keratinocyte differentiation. We demonstrate that caspase-14 expression extended to multiple cell lines derived from simple epithelia of the breast, prostate, and stomach. In keratinocytes and breast epithelial cells, caspase-14 expression was upregulated in high-density cultures and during forced suspension culture. These effects were primarily due to transcriptional activation as indicated by reporter gene assays using a 2 kb caspase-14 promoter fragment. Importantly, caspase-14 was not cleaved during forced suspension culture of either cell type although this treatment induced caspase-dependent apoptosis (anoikis). Forced expression of caspase-14 in immortalized human keratinocytes had no effect on cell death in forced suspension nor was the transfected caspase-14 processed in this setting. In contrast to postconfluent and forced suspension culture, terminal differentiation of keratinocytes induced in vitro by Ca2+ treatment was not associated with increased caspase-14 expression or promoter activity. Our results indicate that (1) caspase-14 is expressed not only in complex but also simple epithelia; (2) cells derived from complex and simple epithelia upregulate caspase-14 expression in conditions of high cell density or lack of matrix interaction and; (3) in both cell types this phenomenon is due to transcriptional regulation.

Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC

Abstract: Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-κB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DED-containing protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast two-hybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD- and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-κB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.

A conserved xiap-interaction motif in caspase-9 and smac/diablo for mediating apoptosis

Abstract: The invention provides caspase-9-related peptides and polypeptides capable of binding to an Inhibitor of Apoptosis Protein (IAP), as well as caspase-9 mutant that fail to undergo normal processing and fail to bind to an IAP. Nucleic acid molecules, including expression vectors, encoding such peptides and polypeptides are also provided. Such peptides and polypeptides, are useful for inducing apoptosis and identifying inhibitors and enhancer of apoptosis.

Omi and domains thereof that disrupt iap-caspase interaction

Abstract: An isolated nucleic acid molecule comprising a polynucleotide having a sequence encoding a peptide or polypeptide of Omi having at least an N-terminus amino acid sequence of Ala-Val-Pro-Ser and up to 321 contiguous amino acid residues that can be derived from residues 138-458 of SEQ ID NO:1 or a functional variant of each, each of which is capable of specifically binding to at least a portion of an Inhibitor of Apoptosis protein. This peptide can be used in a method to modulate apoptosis or to identify modulators of apoptosis as well as in therapeutic uses.

Inhibition of interleukin-1-beta secretion by card proteins

Abstract: The present invention provides isolated Pseudo-ICE and ICE-Like, functional fragments thereof, or immunogenic fragments thereof and nucleic acid molecules encoding the above polypeptides. Also provided are various methods of using these polypeptides or nucleic acid molecules in modulating apoptosis or inflammation.

Control of apoptosis by using small molecule regulators of Bcl-2 family proteins

Abstract: The potent biological activity of CPM-1285 suggests that it may represent a promising lead for the development of new anticancer agents. The cell permeable Bcl-2 inhibitor can also be used as a chemical probe to study the in vivo mechanism and signaling pathway of the Bcl-2 family. Unlike other peptides that are active only in vitro or in the cell-free system, the cell-permeable peptide approach described here provides a new tool to analyze the function of the Bcl-2 family in living cells and animals.

Ipaf, an ice-protease activating factor

Abstract: Ipaf nucleic acid molecules and encoded polypeptides are provided. Also provided are methods of using Ipaf polypeptides and nucleic acid molecules. The Invention also provides methodes of screening for inhibitors or enhancers of Ipaf-mediated apoptosis or procaspase-1 processing. The invention further provides production, isolation and purification of Ipaf polypeptides as well as antibodies against said polypeptides. In addition, the invention provides compositions of Ipaf polypeptides, anti-Ipaf antibodies, and/or Ipaf nucleic acid molecules that can be utilized for diagnostic or therapeutic purposes.

Antibodies to Mch6 polypeptides

Abstract: The invention provides an isolated gene encoding Mch6 as well as functional fragments thereof. Also provided are isolated nucleic acid sequences encoding Mch6 or functional fragments thereof. The gene or nucleic acid sequences can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch6 nucleotide sequences. The invention further provides an isolated Mch6 polypeptide and isolated large and small subunits of the Mch6 polypeptide, including functional fragments thereof.