【作者】 徐珺劼；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2007， 博士

【摘要】 本论文的工作重点包括S．cerevisiae Urm1蛋白的克隆、表达纯化并用核磁共振的方法解析了溶液中Urm1蛋白的结构。基于结构，我们分析研究了修饰蛋白与载硫蛋白的进化关系以及Urm1修饰体系中Urm1与它的活化酶Uba4的相互作用模式。我们作了整个泛素超家族蛋白的结构比较和结构联配的序列比对。进化分析显示，Urm1是泛素超家族的分子化石，它的结构和序列可能最好的保守了这个超家族共同祖先蛋白质的特征。我们还推测了Urm1与Uba4的作用界面。另外，我们还采用蛋白质组学方法来分析Urm1修饰体系的靶蛋白。第一章是蛋白质修饰体系的简介，介绍了蛋白质修饰体系的分类和生物学功能。目前已发现的修饰体系有11类，包括：Ubiquitin、NEDD8／Rub1、SUMO、ISG15／UCRP、FAT10、FUB1、UBL5／Hub1、Atg8 and Atg12、Ufm1和Urm1修饰体系。本章重点介绍了Urm1修饰体系的研究背景。Urm1修饰体系自2000年首次被报道，现在已得到越来越多的关注。已经有报道Urm1修饰体系在酵母的出芽生殖、营养缺陷时的侵染性生长的形态发生中发挥功能、参与TOR信号传导途径下游通路的调控和氧化应激反应的调控。但是目前关于Urm1修饰体系功能的研究还主要是通过缺失基因观察表型，更具体的作用机理和可能还有待进一步的研究发现。本章还介绍了关于修饰蛋白进化起源的研究。第二章介绍了分子进化研究的基本理论和研究方法。分子进化钟与中性理论是分子进化研究的基础。构建进化树是研究分子进化的主要手段。构建序列进化树的步骤包括：1．序列比对建立数据模型。2．决定取代模型。3．选择建树方法。4．进化树搜索。5．确定树根。6．进化树评估。我们还介绍了蛋白质进化的基本特点和蛋白质进化关系的识别。第三章中我们解析了S．cerevisae Urm1蛋白的溶液结构，并分析了其进化意义。从Saccharomyces cerevisae(S288C)菌株染色体中获取编码Urm1蛋白的基因，构建表达菌株，经纯化以及同位素标记的预处理，通过核磁共振技术，在计算机软件辅助下，计算出S．cerevisae Urm1蛋白在水溶液中的结构。Urm1的空间结构是由一个含有五段β-strand的β-sheet和四段α螺旋折叠而成。β-strand以21534的顺序组装成β-sheet曲面。四段螺旋在空间上相互靠近，将β-sheet曲面的凹面封闭，形成一个疏水的核心，包装成紧凑的球形结构。α2螺旋内侧疏水残基与β-sheet凹面疏水残基的疏水相互作用是维持此球形结构域的主要作用力。α1和α3在空间上相互靠近，可以检测到它们的远程NOE。紧随β4 strand后的四个残基折叠成小螺旋α4，可以观察到α2螺旋N末端前的残基(33-35)与α4螺旋C末端后的几个残基(80-83)间的远程NOE。Urm1在C末端的六个残基构成柔性的尾巴从球形结构域伸出。结构相似性比较分析显示，Urm1与ThiS，MoaD的结构相似程度更高于它与其它修饰蛋白的结构相似性。Urm1的表面静电，疏水特性也与MoaD的分子表面特征相似，意味着它们可能以相似的方式与配体蛋白质发生相互作用。序列比对和结构比较的结果共同支持了修饰蛋白质由古老载硫蛋白进化产生的推测，同时为Urm1是泛素超家族中的‘分子化石’，即Urm1最好的保守了泛素超家族的共有祖先的分子特性的推测提供了重要的证据。另外，分子表面性质分析提示，类似MoaD，Urm1通过一个暴露的疏水平台和平台周围一些带电残基与配体发生相互作用。我们相信Urm1蛋白结构的解析为揭示泛素超家族蛋白质进化起源提供了重要证据，同时也为进一步研究修饰蛋白质的生理功能和修饰机理提供了新的信息。第四章介绍了我们用蛋白质组学方法研究Urm1修饰体系靶蛋白的工作进展。首先综述介绍了蛋白质组学概念和研究策略，以及用蛋白质组学方法分析修饰蛋白体系靶蛋白的策略和研究进展。我们希望通过蛋白质组学方法分析鉴定Urm1修饰体系的潜在的靶蛋白，帮助我们更深入的了解Urm1修饰体系的生理功能、作用机理。目前我们已经成功构建了HisX6-Urm1的酵母诱导表达菌株，并建立了初步的纯化富集方案。我们遇到了无法用western blot检测内源表达蛋白及检测不到Urm1化靶蛋白等问题，在下一步的工作中还需要进一步优化我们的纯化富集方案和检测手段，以及用2DLC-MS／MS分析纯化产物。更多还原

【Abstract】 Our work focuses on the cloning, expression, purification and solving the solution structure of S. cerevisiae Urm1 protein by NMR method. Structure of Urm1 is based to infer the evolutionary relationships between the protein modifiers and sulfur carrier proteins and explore the function and interaction pattern of the Urm1 conjugation system. We use both structural comparison and phylogenetic analysis of the ubiquitin superfamily to test the hypothesis that Urm1 is a "molecular fossil" in the superfamily and has the most conserved structural features of the family’s common ancestor. We presumed a possible partner-binding interface of Urm1 to Uba4. Besides, we are studying the targets of Urm1 conjugation system using proteomic strategy and methods.First chapter is the brief review of protein modifiers. Classification of protein modifiers and their biological functions were introduced. Till now, 11 protein conjugation systems have been identified, include: Ubiquitin, NEDD8/Rubl, SUMO, ISG15/UCRP, FAT10, FUB1, UBL5/Hub1, Atg8, Atg12, Ufm1 and Urm1 conjugation systems. We focused on introducing the investigation background of Urm1 conjugation system. The gene for all urmylation pathway proteins, Urm1 and Uba4, is essential for S. cerevisiae viability during budding in vegetative growth and is shown to play a role in invasive growth into agar in the haploid state and in pseudohyphal growth and cell elongation under starvation conditions in the diploid state. A functional cross-link between the TOR (target of rapamycin) signaling pathway and the urmylation pathway was also detected, in which Urm1 was shown to be involved in nutrient sensing. It was found that Urm1 covalently attaches to antioxidant protein Ahp1 to modulate its activity in oxidant-stress response. However, much still remains to be learned about the biological function and functional mechanism of the Urm1 conjugation system. We also introduced the evolutionary study on protein modifiers in this chapter.The second chapter introduced the primary theory and study method on molecular evolution. ’Molecular clock’ and ’Neutral theory’ based the investigation on molecular evolution. Construction of evolutionary and phylogenetic tree is usually used to study molecular evolution. The steps of constructing of evolutionary and phylogenetic tree include: 1. sequence alignment orientated data model. 2. Determination of substitute model. 3. Selective constructing. 4. Query of phylogenetic tree. 5. Root determination. 6. Estimation of phylogenetic tree. We also introduced the characters of protein evolution and identification of protein evolutionary relationships.In the third chapter, we solved the solution structure of S. cerevisae Urm1 and analyzed its evolutionary implications. The gene coding full length Urm1 protein was obtained by PCR from Saccharomyces cerevisae(S288C) genome. Recombinant Urm1 was cloned and expressed in E.coli and was purified using Ni-chelating column. The solution structure of S. cerevisae Urm1 was obtained by heteronuclear three-dimensional spectroscopy and calculated with the supplement of computer softwares. The structure of Urm1 contains one five-strandβ-sheet and fourα-helices. Theβ-sheet is arranged in the order 21534, as in ubiquitin; four helices together back the curvedβ-sheet on the concave side; Interaction between the inner face of theα2-helix and the concave face of theβ-sheet form a hydrophobic core, which is essential to maintain the compact fold. The short one-turnα4-helix follows theβ4-strand tightly. Long-range NOEs were observed from the N terminus (residues 33-35) of a2-helix to the residues (80-83) after the C terminus ofα4-helix. The C terminus (residues 95-99) is flexible and protrudes from the globular fold as a tail. Structural comparison indicates that Urm1 shares higher structural similarity with ThiS and MoaD than with other protein modifiers. The similarities of 3D structure and hydrophobic and electrostatic surface features between Urm1 and MoaD suggest that they may interact with partners in a similar manner. Both structural comparison and phylogenetic analysis of the ubiquitin superfamily, with emphasis on the Urm1 family, indicate that Urm1 is the unique ’molecular fossil’ that has the most conserved structural and sequence features of the common ancestor of the entire superfamily. On the other hand, it is proposed that, analogy to MoaD, the partner-binding interface of Urm1 may consist of a hydrophobic region exposed to solvent and some charged residues. We believe that the solved Urm1 protein structure, which is considered to be a critical piece of evidence for inferring the evolutionary origin of the ubiquitin superfamily, would also be extremely informative for further investigation of the complex function and mechanism of the modification pathway during the evolution of protein modifiers.The fourth chapter introduced the work processed on identifying the modification targets of Urm1 conjugation system. The concepts of proteomics and the study strategy and methods were introduced firstly. Then the strategy for proteomic studies of protein modifiers was discussed. To identify potential targets of Urm1 conjugation system is significant for uncovering the biological process the system involved and its functional mechanism. Till now, we have successfully constructed the yeast strain which can be induced to express His-tagged Urm1 protein. We also have set up a primary strategy for purifying and enriching Urm1 and Urm1 conjugated proteins. However, we can not detect the Urm1 protein expressed by wild-type strains and the Urm1 conjugated proteins seem to be missed during purification according to Western blot results. In the future work, purification strategy and detection method should be optimized and we will try to analysis the purified product by 2DLC-MS/MS method.更多还原