Western blot was used to measure the protein expression of ATXN3 and YAP, while the YAP target genes were measured by real-time PCR. CCK8 assay was used to detect cell viability; transwell invasion assay was used to measure the invasion ability of PC. The xeno-graft tumor model was used for in vivo study. Protein stability assay was used to detect YAP protein degradation. Immuno-precipitation assay was used to detect the interaction domain between YAP and ATXN3. The ubiquitin-based Immuno-precipitation assays were used to detect the specific ubiquitination manner happened on YAP.
In the present study, we identified ATXN3, a DUB enzyme in the ubiquitin-specific proteases family, as a bona fide deubiquitylase of YAP in PC. ATXN3 was shown to interact with, deubiquitylate, and stabilize YAP in a deubiquitylation activity-dependent manner. Depletion of ATXN3 decreased the YAP protein level and the expression of YAP/TEAD target genes in PC, including CTGF, ANKRD1 and CYR61. Further mechanistic study revealed that the Josephin domain of ATXN3 interacted with the WW domain of YAP. ATXN3 stabilized YAP protein via inhibiting K48-specific poly-ubiquitination process on YAP protein. In addition, ATXN3 depletion significantly decreased PC cell proliferation, invasion and stem-like properties. The effects induced by ATXN3 depletion could be rescued by further YAP overexpression.
In general, our findings establish a previously undocumented catalytic role for ATXN3 as a deubiquitinating enzyme of YAP and provides a possible target for the therapy of PC. Video Abstract.
方法:Westernblot检测ATXN3和YAP的蛋白表达,同时通过实时PCR检测YAP靶基因。CCK8试验用于检测细胞活力;transwell侵袭试验用于测量PC的侵袭能力。异种移植物肿瘤模型用于体内研究。蛋白质稳定性测定用于检测YAP蛋白质降解。免疫沉淀法用于检测YAP和ATXN3之间的相互作用域。基于泛素的免疫沉淀实验用于检测YAP上发生的特定泛素化方式。
结果:在本研究中,我们鉴定了ATXN3,泛素特异性蛋白酶家族中的一种DUB酶,作为PC中YAP的真正去泛素酶。ATXN3被证明与,去泛素,并以去泛素化活性依赖性方式稳定YAP。ATXN3的缺失降低了PC中YAP蛋白水平和YAP/TEAD靶基因的表达,包括CTGF,ANKRD1和CYR61。进一步的机理研究表明,ATXN3的Josephin域与YAP的WW域相互作用。ATXN3通过抑制K48特异性聚泛素化过程稳定YAP蛋白。此外,ATXN3耗竭显著降低PC细胞增殖,入侵和茎样特性。由ATXN3耗竭诱导的效应可以通过进一步的YAP过表达来挽救。
结论:一般来说,我们的发现确立了ATXN3作为YAP的去泛素化酶的催化作用,并为PC的治疗提供了可能的靶点.视频摘要。