title: Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.
abstract: Survivin inhibits apoptosis in numerous tumor cell lines and has emerged as promising target for cancer therapy. The anti-apoptotic effect of survivin was attributed to a direct interaction with XIAP (X-linked inhibitor of apoptosis) and to an indirect effect, where survivin antagonizes the anti-XIAP action of Smac. The direct interaction is thought to lead to synergistic inhibition of caspase-9 and, at the same time, to enhanced stability of XIAP by reducing its auto-ubiquitination. Using recombinant proteins, we have investigated the influence of survivin on the inhibition of caspase-9 by XIAP in vitro. With a fluorescence-based assay for the apoptosome-stimulated activity of caspase-9, we show that survivin has no effect on the inhibition of caspase-9 by XIAP, neither in the presence nor in the absence of Smac. Employing analytical size exclusion chromatography (SEC) and analytical ultracentrifugation, we show that survivin does not physically interact with XIAP. We confirm in vitro that XIAP ubiquitinates itself in the presence of the appropriate recombinant enzymes and Mg2+-ATP and could show that survivin neither influences the kinetics nor the extent of XIAP's self-ubiquitination. Our results call for a revision of the current view of how survivin interferes with the mitochondrial pathway of apoptosis.
authors.affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors.forename: Franziska K
authors.initials: FK
authors.lastname: Zumbrägel
authors.affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors.forename: Dominik A
authors.initials: DA
authors.lastname: Machtens
authors.affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Curth
authors.affiliation: Georg-August-University, Institute for Medical Microbiology, Kreuzbergring 57, 37075 Göttingen, Germany.
authors.forename: Carsten G K
authors.initials: CGK
authors.lastname: Lüder
authors.affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Reubold
authors.affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Electronic address: Eschenburg.Susanne@mh-hannover.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Eschenburg
mesh_terms: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053453:Caspase 9 / Q000378:metabolism*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D000077022:Survivin; D054875:Ubiquitination; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C107489:BIRC5 protein, human; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D000077022:Survivin; D051636:X-Linked Inhibitor of Apoptosis Protein; D053453:Caspase 9
keywords: Apoptosis; Caspase inhibition; Smac; Survivin; Ubiquitination; XIAP