嗜铬细胞瘤蛋白质琥珀酰化的研究——鉴定，功能与机制

Succinylation of lysine residues is a newly-discovered, widespread post-translational modification (PTM), but its function in physiology and pathology were uncleared. Evidence suggested that the protein succinylation is associated with phaeochromocytoma syndrome (PCC), and two tumor inhibitor genes, SDH and PCC related protein (PCCRP) could affect the protein succinylation in PCC. This proposal would target the critical issues of protein succinylation in PCC, take full advantage of proteomics technology, systematically identify the protein succinylation in PCC and study their functions in the tumor development. In this proposal, firstly, succinylation proteins in PCC cell lines would be enriched by immunogenicity enrichment at peptide level, and analyzed by mass spectrum. The affection of tumor suppresser gene SDH and PCCRP to the succinylation proteome in PCC cells would be quantitatively studied. Secondly, by Bioinformatics analysis and RNAi screen, the PCCRP mediated nuclear localization succinylated tumor suppressor proteins would be discovered. Thirdly, the regulation mechanism of protein succinylation of by SDH and PCCRP would be studied. Lastly, the relationship between protein succinylation and tumor development in PCC would be studied, and whether PCCRP suppress PCC tumor through regulation of protein succinylation of some crucial proteins or not would be explored. We hope that the implementation of this proposal would bridge the protein succinylation and tumor development for the first time, and uncover a new molecular mechanism for PCC development, which could provide theoretical guide for the diagnosis and treatment of PCC.

赖氨酸琥珀酰化是一种近年来发现的广泛存在的翻译后修饰，然而它在细胞中生理病理功能尚不清楚。已有线索表明蛋白琥珀酰化与嗜铬细胞瘤（PCC）相关，且肿瘤抑制基因SDH和PCCRP可以影响PCC琥珀酰化。本项目拟针对PCC琥珀酰化蛋白等关键问题，充分利用蛋白质组学的特点和优势，系统鉴定PCC琥珀酰化蛋白并研究其在肿瘤的发生中的作用。本研究首先利用免疫亲和方法富集PCC细胞中琥珀酰化蛋白，通过质谱技术蛋白鉴定并定量研究SDH和PCCRP对PCC琥珀酰化蛋白的影响。第二，通过生物信息学分析和RNAi筛选筛选PCCRP介导的核定位抑癌琥珀酰化蛋白。第三研究SDH和PCCRP影响蛋白琥珀酰化的机制。最后研究PCC中蛋白琥珀酰化与肿瘤发生的关系以及PCCRP是否通过影响某些关键蛋白琥珀酰化抑制肿瘤。本项目首次揭示赖氨酸琥珀酰化与肿瘤发生的关系，并发现PCC发生的新分子机制，为PCC诊治提供理论依据和指导

赖氨酸琥珀酰化修饰是一种近年来新发现的广泛存在的翻译后修饰，然而它在细胞中生理病理功能尚不清楚。我们发现，蛋白琥珀酰化与嗜铬细胞瘤（PCC）相关，且肿瘤抑制基因SDH和PCCRP(即GIPC2)可以影响细胞蛋白琥珀酰化。本研究首先利用多肽水平免疫亲和富集方法富集PCC细胞中琥珀酰化蛋白，通过SILAC定量技术鉴定并定量研究293T细胞，DMS（一种SDH抑制剂）处理的293T细胞和DMS+PCCRP处理的293T细胞的琥珀酰化的变化。我们鉴定6003个总蛋白，琥珀酰化修饰蛋白257个，585个琥珀酰化修饰位点。我们发现124个琥珀酰化位点（91个蛋白）在DMS刺激下表达上调，而这些蛋白的琥珀酰化增加可被PCCRP恢复和逆转。通过机制研究，我们发现我们SDHx失活通过下调PCCRP激活pErK1/2来抑制p27从而促进嗜铬细胞瘤的增殖。SDH和PCCRP不是依靠Sirt5调节细胞内的琥珀酰化。我们筛选并验证了19个与PCCRP相互作用的核定位蛋白。我们验证了肿瘤相关蛋白HSP90AB1在DMS作用下琥珀酰化程度增加且该过程被PCCRP逆转。HSP90AB1还同PCCRP相互作用。PCCRP调控的HSP90AB1去琥珀酰化可能是嗜铬细胞瘤发生的重要机制。本课题为研究SDH和PCCRP调控嗜铬细胞瘤发生的机制提供了重要基础。