Structural determinants of caspase-9 inhibition by the vaccinia virus protein, F1L.
ABSTRACT In multicellular organisms, apoptosis is a powerful method of host defense against viral infection. Apoptosis is mediated by a cascade of caspase-family proteases that commit infected cells to a form of programmed cell death. Therefore, to replicate within host cells, viruses have developed various strategies to inhibit caspase activation. In the mitochondrial cell-death pathway, release of cytochrome c from mitochondria into the cytosol triggers assembly of the oligomeric apoptosome, resulting in dimerization and activation of the apical caspase-9 (C9), and in turn its downstream effector caspases, leading to apoptosis. We previously showed that the vaccinia virus-encoded Bcl-2-like protein, F1L, which suppresses cytochrome c release by binding Bcl-2 family proteins, is also a C9 inhibitor. Here, we identify a novel motif within the flexible N-terminal region of F1L that is necessary and sufficient for interaction with and inhibition of C9. Based on functional studies and mutagenesis, we developed an atomic model of the complex in which F1L inhibits C9 by engaging the active site in the reverse orientation with respect to substrate peptides, in a manner analogous to that of XIAP-mediated inhibition of caspases-3 and -7. These studies offer new insights into the mechanism of apoptosome inhibition by F1L as well as novel probes to understand the molecular bases of apoptosome regulation and turnover. They also suggest how the two distinct functionalities of F1L (inhibition of C9 and suppression of pro-apoptotic Bcl-2 family proteins) may operate in a cellular setting.
Article: XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs.[show abstract] [hide abstract]
ABSTRACT: The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.The EMBO Journal 03/2005; 24(3):645-55. · 9.20 Impact Factor
Article: [Diagnosis and significance of minimal residual disease in patients with colorectal carcinoma].[show abstract] [hide abstract]
ABSTRACT: Progression of malignant tumours to metastatic disease after surgery in curative intent is due to usually few and with conventional diagnostic techniques undetectable residual malignant cells. These cells must have been spread either before or during surgery. The more sensitive and specific detection of these malignant cells in various clinically relevant compartments of the patients organism became possible through new immunocytochemical and molecular tools. Various clinical studies underline the strong prognostic impact of the detection of disseminated cancer cells. However, many technical aspects did not yet allow sufficient standardization of these procedures and led to significantly varying results among different scientific groups. It is one of our primary aims to test, whether the detection of disseminated cancer cells might guide the indication for adjuvant therapies and might predict the efficacy of therapies in patients with solid cancers.Zentralblatt für Chirurgie 02/2000; 125 Suppl 1:15-9. · 1.02 Impact Factor
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ABSTRACT: Although nurses have the major responsibility for pain management, little is known about nurses' responses to patients in the process of managing acute pain. To examine the relationship between nurses' empathic responses and their patients' pain intensity and analgesic administration after surgery. Two hundred twenty-five patients from four cardiovascular units in three university-affiliated hospitals were interviewed on the third day after their initial, uncomplicated coronary artery bypass graft (CABG) surgery about their pain and current pain management. Concurrently, their nurses' (n = 94) empathy and pain knowledge and beliefs were assessed. Patient data were aggregated and linked with the assigned nurse to form 80 nurse-patient pairs. Nurses were moderately empathic, and their responses did not significantly influence their patients' pain intensity or analgesia administered. Patients reported moderate to severe pain but received only 47% of their prescribed analgesia. Patients' perceptions of their nurse's attention to their pain were not positive, and empathy explained only 3% of variance in patients' pain intensity. Deficits in knowledge and misbeliefs about pain management were evident for nurses independent of empathy, and knowledge explained 7% of variance in analgesia administered. Hospital sites varied significantly in analgesic practices and pain inservice education for nurses. Empathy was not associated with patients' pain intensity or analgesic administration.Nursing Research 49(4):191-200. · 1.40 Impact Factor