title: Novel small molecules relieve prothymosin alpha-mediated inhibition of apoptosome formation by blocking its interaction with Apaf-1.
abstract: Structurally diverse small molecules, including 5-(2-benzofuryl)-4-phenyl-1,2,4-triazole-3-thiol (BETT), have been identified via high-throughput screening as activators of caspase-3 in HeLa cell extracts. However, little is known about their mechanism of action. In this study, we investigate how BETT regulates prothymosin alpha (ProT), a nuclear protein previously shown to play essential roles in apoptosis. We first showed that Apaf-1 is the direct target protein of BETT. We further demonstrated that BETT relieved ProT-mediated inhibition of apoptosome formation by blocking the interaction between Apaf-1 and ProT. Using two-dimensional (1)H-(15)N heteronuclear single-quantum correlation (HSQC) experiments, we were also able to examine the interaction between Apaf-1 and (15)N-labeled ProT alpha. Furthermore, we were able to reconstitute the entire caspase-3 activation pathway using purified ProT, Apaf-1, procaspase-9, procaspase-3, Hsp70, cytochrome c, PHAPI, CAS, and regulatory compounds to mimic stress-induced apoptosis in vitro. Together, these studies would lead to novel and specific methods for the prevention, diagnosis, and treatment of human cancer.
authors.affiliation: Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. xqi@scripps.edu
authors.forename: Xin
authors.initials: X
authors.lastname: Qi
authors.forename: Lai
authors.initials: L
authors.lastname: Wang
authors.forename: Fenghe
authors.initials: F
authors.lastname: Du
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053478:Apoptosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D053477:Apoptotic Protease-Activating Factor 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D011498:Protein Precursors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D013947:Thymosin / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014230:Triazoles / Q000494:pharmacology*
chemical_list: C548686:5-(2-benzofuryl)-4-phenyl-1,2,4-triazole-3-thiol; C506592:APAF1 protein, human; D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; D061945:Caspase Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; D014230:Triazoles; C040782:prothymosin alpha; D013947:Thymosin; D053148:Caspase 3