Intracellular beta-tubulin/chaperonin containing TCP1-beta complex serves as a novel chemotherapeutic target against drug-resistant tumors.
In the present study, treatment of HEK-293 cells with the synthetic small molecule N-iodoacetyl-tryptophan (I-Trp) at submicromolar concentrations efficiently induced cell apoptosis as judged from the accumulation of sub-G(0) cells and intracellular DNA fragmentation. Activation of all intracellular caspases, except caspase-1, was detected in I-Trp-treated cells. Proteomic analysis revealed that beta-tubulin acted as a specific intracellular target of I-Trp. Protein fingerprinting analysis indicated that the Cys(354) residue in the peptide fragment TAVCDIPPR of beta-tubulin, which is located at the binding interface with chaperonin containing TCP1-beta (CCT-beta), was alkylated by I-Trp. Moreover, site-directed mutagenesis of Cys(354) (Cys-Ala) abolished the incorporation of I-Trp into beta-tubulin, suggesting Cys(354) is indeed the targeting site of I-Trp. Immunoprecipitation showed that the beta-tubulin/CCT-beta complex was constitutively formed but disrupted after treatment with I-Trp. Overexpression of the truncated beta-tubulin (T351-S364) or treatment with I-Trp or the synthetic peptide Myr-TAVCDIPPRG caused more severe cell apoptosis in multidrug-resistant MES-SA/Dx5 cancer cells due to higher levels of CCT-beta relative to wild-type MES-SA cancer cells. Silencing the expression of CCT-beta rendered MES-SA/Dx5 cells less sensitive to I-Trp-induced apoptotic cell death. These findings suggest that the beta-tubulin/CCT-beta complex may serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-beta-overexpressing tumors.
Lin
Yuan-Feng
YF
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan ROC.
Tsai
Wen-Ping
WP
Liu
Hon-Ge
HG
Liang
Po-Huang
PH
D000478:Alkylation; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites / Q000235:genetics; D020170:Caspase 1 / Q000378:metabolism; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism*; D003545:Cysteine; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C533298:CCT2 protein, human; D010446:Peptide Fragments; C533304:TCP1 protein, human; D014404:Tubulin; D014364:Tryptophan; D020170:Caspase 1; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins; D003545:Cysteine