Ubiquitin修饰的蛋白质组学分析及HDAC5的Sumo修饰机制研究

【作者】 谭锋维；

【导师】 强伯勤； 罗楹； 袁建刚； 彭小忠；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 Ubiquitin是一个由76个氨基酸残基组成的广泛并且只存在于真核细胞的多肽分子。Ubiquitination最重要的功能是介导底物蛋白通过26S蛋白酶体快速降解。Ubiquitin通过修饰各种重要的功能蛋白，精确地调节着细胞周期、细胞增殖与分化、细胞凋亡等生理过程。在真核细胞内，还存在类似于Ubiquitin的小分子多肽，称为UBLs(Ubiquitin-like modifiers)。Sumo(Small ubiquitin-like modifier)是研究得最为深入的一种UBLs。Sumo具有与Ubiquitin相类似的三维空间结构，以Ubiquitination类似的方式对相应的底物蛋白进行修饰。Sumos修饰不介导蛋白质降解，而是通过改变细胞定位、蛋白活性、蛋白之间相互作用等不同的机制在细胞的各种生理活动中发挥重要的调节作用。在本文中，我们从组学水平对肝细胞蛋白质的Ubiquitination进行了分析，并且研究了HDAC5的Sumo修饰机制。我们利用蛋白质组学的研究手段对肝细胞内Ubiquitin底物蛋白进行了纯化分离，并对分离出的Ubiquitin底物蛋白进行了质谱鉴定。S5a是26S蛋白酶体的Ubiquitin识别亚基，能够通过两个独立的UIMs(Ubiquitin-interacting motifs)特异性结合发生Polyubiquitination的蛋白。我们利用S5a亲和层析从人正常肝细胞(Chang’liver cell line)中纯化了发生Polyubqiquitination的蛋白，并且使用高通量质谱方法LC／LC-MS／MS (Multidimensional chromatography coupled with tandem mass spectrometry)进行了蛋白质成分鉴定。通过上述实验，我们一共确定了81个Polyubiquitination的底物蛋白，同时通过数据分析预测了分布于17个蛋白中的19个可能的Ubiquitin特异性修饰位点。与此同时，我们利用生物化学的方法对Sumolation的修饰机制进行了研究。我们鉴定了一个新的Sumo底物蛋白——组蛋白去乙酰化酶5(HDAC5)，并深入研究了Sumolation的修饰机制。组蛋白去乙酰化酶(Histone deacetylases，HDACs)通过改变组蛋白的乙酰化状态改变着染色质的结构，是十分重要的基因表达调节因子。HDAC4是Sumo的重要底物之一，我们通过体内Sumoylation分析实验发现了与HDAC4同属Ⅱa家族的HDAC5也能够被Sumo修饰。含有SP-RING结构域的PIASs(Protein inhibitorsof activated STAT proteins)家族蛋白是目前发现的最主要的Sumo E3连接酶，在基因的转录调节中发挥重要作用。我们通过免疫沉淀实验证明TPIASs和HDAC5在细胞内相互作用。将不同的PIASs成员加入HDAC5的Sumoylation体内分析系统，进一步发现PIAS1特异性促进Sumo1对HDAC5的修饰，而PIASy特异性促进Sumo2对HDAC5的修饰。我们的工作也发现在HDAC5中符合ψKXE的赖氨酸(K606)为Sumo2和Sumo3的特异性修饰位点。根据文献报道并结合本研究所得实验结果，我们推测第600氨基酸附近的区域为Ⅱa类HDACs保守的Sumolation修饰位置。更多还原

【Abstract】 Ubiquitin is a highly conserved 76-amino acid polypeptide expressed in all eukaryotes but absent in bacteria and archaea. Polyubiquitin through Lys48 linkage targets modified proteins for ATP-dependent degradation by the 26S proteasome. Ubiquitination plays an essential role in cellular functions such as cell cycle progressing, cellular proliferation and differentiation, apoptosis, etc. UBLs (ubiquitin-like modifers) that share similarities with ubiquitin as a post- translational protein modifier have been identified in eukaryotic cells. The most well studied member of this ubiquitin-like family is Sumo (Small Ubiquitin-like modifier). With an ubiquitin-like 3D structure, Sumo is conjugated to proteins via a similar mechanism as ubiquitin. Unlike ubiquitination, which usually targets proteins to degaradation, sumolation mediates a number of cellular processes by regulate the behaviors of substrate proteins such as subcellular localization, enzymatic activity, protein-protein interaction. Here, we studied the ubiquitination in liver cells by proteomic approach and the sumolation of HDAC5 respectively.We purified and identified the ubiquitinated proteins by proteomics-based approaches. The S5a subunit of 26S proteasome was originally identified as a protein capable of binding poly ubiquitin chains through Lys48 linkage. 81 putative polyubiquitinated proteins in normal liver cells (Chang’ liver cell) were identified in this study using S5a-affinity chromatography coupled LC/LC-MS/MS analysis. Moreover, 19 putative ubiquitination sites were identified by mass spectrometry.Moreover, we show that HDAC5 is sumolated in vivo and this modification is regulated by PIASs. By deacetylating histone and modifying chramotin structure, HDACs (Histone Deacetylase) play essential roles in regulating gene expression. Previous research showed that HDAC4 is modified by sumo, we found that HDAC5 is a new substrate of sumo by in vivo sumoylation assays in this report. PIASs (Protein Inhibitors of Activated STAT Protein), which contain a conserved SP-RING domain, are considered as the common E3 ligase in sumolation pathway. After confirming the interaction between PIASs and HDAC5 by co-immunoprecipitation, we found that PIAS1 enhances the Sumo1-modification of HDAC5, while PIASy enhances the Sumo2-modification of HDAC5 specifically. Our data also showed that K606 on HDAC5, which matches a consensus sumoylation motif, is a specifical Sumo2/3 modification site. Bioinforrnatic analysis leads us to assume that the sumolation on conserved region (about the 600 amino acid from the N-terminus) is a common characteristic of ClassⅡa HDACs.更多还原