Bhattacharya Seemana, Ghosh Mrinal K
Signal Transduction in Cancer and Stem Cells Laboratory, Division of Cancer Biology and Inflammatory Disorder, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata, 700 032, India.
Cell Oncol (Dordr). 2015 Aug;38(4):265-77. doi: 10.1007/s13402-015-0228-6. Epub 2015 Apr 30.
The de-ubiquitinase HAUSP has been reported to exhibit various biological roles implicated in the development of cancer and other pathologies. The dual nature of HAUSP (i.e., oncogenic and tumor suppressive) makes the protein even more versatile. The major aims of this study were to reveal the effect of HAUSP over-expression on the overall proteome and to identify bona fide substrates of HAUSP. In addition, we aimed to unravel the functionality and physiological relevance of the de-ubiquitinating activity of HAUSP on one of its newly identified substrates, TRRAP.
An overall proteome analysis was performed after exogenous HAUSP over-expression in HEK293 cells, followed by 2-dimensional gel electrophoresis (2-DE). Interacting proteins were subsequently isolated using immunoprecipitation and 1-dimensional gel electrophoresis (1-DE). Both were followed by tandem MALDI-TOF/TOF mass spectrometry and gene ontology-based analyses. To validate the functionality of one of the identified substrates (TRRAP), Western blotting, immunocytochemistry, immunoprecipitation, in vivo de-ubiquitination, quantitative real-time PCR and luciferase assays were performed.
The substrate screening indicated that HAUSP may be involved in tumorigenesis, cytoskeletal organization and transport, and chaperone systems. One candidate substrate, TRRAP, was found to physically interact and co-localize with HAUSP. As TRRAP regulates c-MYC expression, and in order to validate the effect of HAUSP on TRRAP, c-MYC protein and mRNA expression levels were analyzed after exogenous HAUSP over-expression. Both were found to be up-regulated. We also found that c-MYC transactivation increased upon exogenous HAUSP over-expression. By using a luciferase reporter assay, we found that a c-MYC responsive promoter exhibited increased activity, which was subsequently abrogated upon TRRAP knockdown.
From our results we conclude that HAUSP may act as an oncogenic protein that can modulate c-MYC expression via TRRAP. Our results provide a new context in which HAUSP may play a role in cancer cell signalling.
去泛素化酶HAUSP已被报道具有多种与癌症及其他病理发展相关的生物学作用。HAUSP的双重性质（即致癌和肿瘤抑制）使该蛋白更加多功能。本研究的主要目的是揭示HAUSP过表达对整体蛋白质组的影响，并鉴定HAUSP的真正底物。此外，我们旨在阐明HAUSP对其新鉴定的底物之一TRRAP的去泛素化活性的功能及生理相关性。
在HEK293细胞中外源过表达HAUSP后进行整体蛋白质组分析，随后进行二维凝胶电泳（2-DE）。随后使用免疫沉淀和一维凝胶电泳（1-DE）分离相互作用的蛋白质。两者均接着进行串联MALDI-TOF/TOF质谱分析和基于基因本体论的分析。为验证所鉴定底物之一（TRRAP）的功能，进行了蛋白质印迹、免疫细胞化学、免疫沉淀、体内去泛素化、定量实时PCR和荧光素酶测定。
底物筛选表明HAUSP可能参与肿瘤发生、细胞骨架组织和运输以及伴侣系统。发现一个候选底物TRRAP与HAUSP发生物理相互作用并共定位。由于TRRAP调节c-MYC表达，并且为了验证HAUSP对TRRAP的影响，在HAUSP外源过表达后分析了c-MYC蛋白和mRNA表达水平。两者均被发现上调。我们还发现外源过表达HAUSP后c-MYC反式激活增加。通过使用荧光素酶报告基因测定，我们发现一个c-MYC反应性启动子活性增加，而在TRRAP敲低后该活性随后被消除。
从我们的结果中我们得出结论，HAUSP可能作为一种致癌蛋白，可通过TRRAP调节c-MYC表达。我们的结果提供了一个新的背景，其中HAUSP可能在癌细胞信号传导中发挥作用。
