title: Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation.
abstract: In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.
authors.affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
authors.forename: Honghao
authors.initials: H
authors.lastname: Zhang
authors.forename: Raghu
authors.initials: R
authors.lastname: Gogada
authors.forename: Neelu
authors.initials: N
authors.lastname: Yadav
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Lella
authors.forename: Mark
authors.initials: M
authors.lastname: Badeaux
authors.forename: Mary
authors.initials: M
authors.lastname: Ayres
authors.forename: Varsha
authors.initials: V
authors.lastname: Gandhi
authors.forename: Dean G
authors.initials: DG
authors.lastname: Tang
authors.forename: Dhyan
authors.initials: D
authors.lastname: Chandra
mesh_terms: D000255:Adenosine Triphosphate / Q000172:deficiency; D053478:Apoptosomes / Q000378:metabolism*; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol; D005347:Fibroblasts; D006801:Humans; D009711:Nucleotides / Q000172:deficiency*; D011485:Protein Binding
chemical_list: D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; D009711:Nucleotides; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D053453:Caspase 9; D020169:Caspases