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E W Humke

Publications and source records attributed to E W Humke.

7 recordsLinked to original sources
PubMed2001

The PYRIN domain: a member of the death domain-fold superfamily.

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.

Amino Acid Sequence↗
PubMed2000

ICEBERG: a novel inhibitor of interleukin-1beta generation.

ProIL-1beta is a proinflammatory cytokine that is proteolytically processed to its active form by caspase-1. Upon receipt of a proinflammatory stimulus, an upstream adaptor, RIP2, binds and oligomerizes caspase-1 zymogen, promoting its autoactivation. ICEBERG is a novel protein that inhibits generation of IL-1beta by interacting with caspase-1 and preventing its association with RIP2. ICEBERG is induced by proinflammatory stimuli, suggesting that it may be part of a negative feedback loop. Consistent with this, enforced retroviral expression of ICEBERG inhibits lipopolysaccharide-induced IL-1beta generation. The structure of ICEBERG reveals it to be a member of the death-domain-fold superfamily. The distribution of surface charge is complementary to the homologous prodomain of caspase-1, suggesting that charge-charge interactions mediate binding of ICEBERG to the prodomain of caspase-1.

Amino Acid Sequence↗
PubMed1999

Caspase-9 can be activated without proteolytic processing.

The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coli is processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP. Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms. To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen. These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors. The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells. Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity.

Apoptosis↗
PubMed1998

ERICE, a novel FLICE-activatable caspase.

Programmed cell death, or apoptosis, is a process of fundamental importance to cellular homeostasis in metazoan organisms (Ellis, R. E., Yuan, J., and Horvitz, H. R. (1991) Annu. Rev. Cell Biol. 7, 663-698). The caspase family of mammalian proteases, related to the nematode death protein CED-3, plays a crucial role in apoptosis and inflammation. We report here the isolation and characterization of a new caspase, tentatively termed ERICE (Evolutionarily Related Interleukin-1beta Converting Enzyme). Based on phylogenetic analysis, ERICE (caspase-13) is a member of the ICE subfamily of caspases which includes caspase-1 (ICE), caspase-4 (ICErel-II, TX, ICH-2), and caspase-5 (ICErel-III, TY). Overexpression of ERICE induces apoptosis of 293 human embryonic kidney cells and MCF7 breast carcinoma cells. Like other members of the subfamily, ERICE is not activated by the serine protease granzyme B, a caspase-activating component of cytotoxic T cell granules. Therefore, ERICE most likely does not play a role in granzyme B-induced cell death. ERICE, however, was activated by caspase-8 (FLICE, MACH, Mch-5), the apical caspase activated upon engagement of death receptors belonging to the tumor necrosis factor family. This is consistent with a potential role for ERICE in this receptor-initiated death pathway.

Amino Acid Sequence↗
PubMed1998

Activation of caspases triggered by cytochrome c in vitro.

Previous studies have shown that Apaf-1 and caspase-9 in the presence of cytochrome c and dATP can form an initiating complex for an apoptotic protease cascade. We have developed a cytochrome c-dependent in vitro system in which caspases downstream of this initiation complex are activated. The activation of caspase-9 from zymogen form to active dimeric protease requires intrinsic enzymatic activity. In contrast, caspase-3 and caspase-7 zymogens are proteolytically processed by active caspase-9. Activation of the above caspases is blocked by a dominant negative form of caspase-9. The in vitro system displays surprising specificity in that other caspases, including 1, 2, 4, 8, 10, and 13, are not activated.

Animals↗
PubMed1994

Modulation of the PC12 cell response to nerve growth factor by antisense oligonucleotide to amyloid precursor protein.

1. The amyloid precursor protein (APP) is widely distributed among eukaryotic cells, however, its precise role in cellular functioning is not fully clarified. APP is glycoprotein membrane constituent and it may facilitate membrane associated functions. 2. The aim of the present study was to examine the possibility that APP may play a role in mediating cellular trophic responses. The methods made use of an antisense oligonucleotide that was prepared to the 5' terminus of APP and shown specifically to reduce the level of APP isoforms. 3. In sequential mixing experiments it was observed that the APP antisense oligonucleotide did not significantly modify the trophic response of PC12 cells pretreated with nerve growth factor (NGF). However, pretreatment of cells with the antisense oligonucleotide diminished NGF-induced increases in cellular size and neurite length. 4. These observations suggest that APP may play a role in modulating the trophic response. The combined use of APP antisense oligonucleotides and neurotrophic agents may find clinical utility in the treatment of Alzheimer-type dementia since it is known that NGF normally causes increases in APP levels.

Alzheimer Disease↗