The death-fold superfamily of homotypic interaction motifs.

The death-fold superfamily encompasses four structurally homologous subfamilies that engage in homotypic, subfamily-restricted interactions. The Death Domains (DDs), the Death Effector Domains (DEDs), the CAspase Recruitment Domains (CARDs) and the PYrin Domains (PYDs) constitute key building blocks involved in the assembly of multimeric complexes implicated in signaling cascades leading to inflammation and cell death. We review the molecular basis of these homotypic domain-domain interactions in light of their structure, function and evolution. In addition, we elaborate on three distinct types of asymmetric interactions that were recently identified from the crystal structures of three multimeric, death-fold complexes: the MyDDosome, the PIDDosome and the Fas/FADD-DISC. Insights into the mechanisms of interaction of death-fold domains will be useful to design strategies for specific modulation of complex formation and might lead to novel therapeutic applications.

The mitochondrial serine protease HtrA2/Omi cleaves RIP1 during apoptosis of Ba/F3 cells induced by growth factor withdrawal.

Interleukin-3 (IL-3) deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 (Bcl-2) overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzoxy-valyl-analyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk). IL-3 withdrawal causes receptor-interacting protein (RIP)1 cleavage into C-terminal fragments of 30 and 25 kDa, and only cleavage leading to the former was prevented by zVAD-fmk. siRNA experiments demonstrated that generation of the 25-kDa fragment was due to a Bcl-2-modulated release of the mitochondrial serine protease high temperature requirement protein A2 (HtrA2)/Omi. Accordingly, recombinant HtrA2/Omi efficiently cleaved mouse RIP1 in vitro, generating fragments matching those observed in IL-3-deprived Ba/F3 cells. The HtrA2/Omi cleavage site in mouse RIP1 was mapped to the intermediate domain and the corresponding N- and C-terminal fragments were impaired in their ability to activate nuclear factor-kappaB, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Interestingly, knockdown of HtrA2/Omi afforded protection against IL-3 withdrawal-induced death in the presence of zVAD-fmk, demonstrating a role for HtrA2/Omi in caspase-independent cell death during growth factor withdrawal by cleaving RIP1.

Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6-P5' undecapeptide retained complete specificity for caspase-7. The corresponding P6-P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P' residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2', and P3' residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

SitePredicting the cleavage of proteinase substrates.

Proteinases are enzymes that play important roles in vital cellular and extracellular processes by hydrolytically cleaving peptide bonds in their protein substrates. This cleavage can be non-specific as part of degradation during protein catabolism or highly specific as part of proteolytic cascades and signal transduction events. Several web tools are available for predicting possible cleavage sites in candidate substrates. Here, we compare existing prediction tools with SitePrediction, a novel and user-friendly tool for identifying potential cleavage sites. This prediction is based on known datasets found in the literature, stored in web-accessible repositories or generated by our own experiments. Comparison of the different programs shows that SitePrediction makes it possible to derive more reliable predictions. In addition, this tool allows the use of a wide range of proteinases.