瑞氏木霉TrUBC介导的泛素化修饰在纤维素酶基因转录调控中的作用机制

Precise control of gene expression is key for cells to carry out various processes as well as to rapidly respond to even subtle environmental changes. One of the efficient ways to fine-tune the expression of a specific gene is to dynamically modulate the function as well as the temporal-spatial status of regulators through post-translational modifications including ubiquitylation. Filamentous fungi Trichoderma reesei is characterized by its outstanding ability to rapidly initiate the launching of cellulase biosynthesis in the presence of cellulose substrates which otherwise are kept silent. Unfortunately, many facets of the precise mechanism involved in the transcription regulation is not clear yet. The present application aims to focus on our recently identified ubiquitin-conjugating enzyme TrUBC, which was shown to play an important role in the induced cellulase gene expression. Systematic studies will be carried out to elucidate the mechanism by which the TrUBC-mediated ubiquitylation participates in the regulation of cellulase gene expression. Attempts made will include the revelation of the dynamic behavior of TrUBC during the induced expression of cellulases, the screening and identification of the coupled ubiquitin ligase E3 as well as the finally modified regulator(s). No efforts will then be spared to thoroughly dissect the mode of ubiquitylation occurring on the substrate including the modification sites, the type of linkage of ubiquitin chains attached to targeted substrates. The effect of the changes in the ubiquitylating machinery and in the modification mode on the final induced expression of cellulase genes will be simultaneously analyzed. The results are anticipated not only to provide deeper insights into the regulation system for T. reesei cellulases, but also to extend the mechanistic understanding on the role of post-translational protein modification in eukaryotic gene regualtion.

基因表达的精确调控是细胞行使正常功能、快速响应外界环境变化的一个重要保证。包括泛素化修饰在内的蛋白质翻译后修饰是一种可通过调节特定因子功能及时空状态而实现对基因表达进行准确调控的有效方式。丝状真菌瑞氏木霉具有在纤维素存在条件下快速合成大量纤维素酶的重要生物学特性，但其转录调控分子机制的许多精细环节并不清楚。本项申请拟围绕前期工作中鉴定到的在纤维素酶基因表达调控中发挥重要作用的泛素结合酶TrUBC，系统研究其介导的泛素化修饰在纤维素酶基因转录调控中的作用机制，包括揭示TrUBC在纤维素酶基因表达调控中的动态作用模式，筛选鉴定与TrUBC偶联的泛素连接酶E3及泛素化修饰的靶点因子，解析靶点因子发生的泛素化修饰模式及该修饰模式对纤维素酶基因转录表达的调控机制。研究结果将不仅有助于全面深化对瑞氏木霉纤维素酶调控体系的认识，还将拓展蛋白翻译后修饰在真核基因表达调控中的作用研究。

丝状真菌里氏木霉（Trichoderma reesei）在纤维素等底物存在的条件下可以快速分泌大量的纤维素酶以实现对纤维素的高效降解。里氏木霉纤维素酶基因的转录表达受到一系列调控因子的控制，其中Xyr1是最关键的转录激活因子，但是其精确的调控机制依然不清楚。本项目围绕泛素结合酶TrUbc4以及与其功能相关的E3TrRsp5在纤维素酶基因转录调控中的作用机制展开系统研究，主要获得了以下研究结果：1）TrUbc4与Xyr1共定位于细胞核，其缺失严重降低纤维素酶基因的表达；2）突变体回补实验发现，TrUbc4的催化活性位点突变体C85A及双突变体F62A/A96D不能回补缺失菌对纤维素酶利用的表型，暗示TrUbc4功能的发挥依赖于E3/E2~Ub功能体，同时N77S突变体的引入表明与TrUbc4功能关联的E3主要是HECT结构域的E3；3）TrUbc4缺失基本不影响xyr1的转录，却严重降低了Xyr1在纤维素酶基因启动子区的占据，这种降低不能通过提高Xyr1的表达量来恢复；4）泛素修饰组学初步分析TrUbc4可能参与里氏木霉多种生理调控过程；5）HECT 结构域E3 TrRsp5在纤维素酶表达过程中发挥着重要的作用，并且催化活性位点784C直接参与纤维素酶基因的表达；6）体外泛素修饰实验中，TrRsp5具有自我泛素化修饰活性，其修饰依赖于TrUbc4与催化活性位点784C；7）TrRsp5缺失基本摧毁了Xyr1在纤维素酶基因启动子区的占据但是却基本不影响Xyr1的转录及表达，这种占据的降低不能通过提高Xyr1的转录水平来恢复。以上结果表明，TrUbc4-TrRsp5泛素功能体通过调控Xyr1在纤维素酶基因启动子区的占据来调节纤维素酶的表达。上述创新结果极大地丰富了纤维素酶基因表达调控机制的研究，并且能够为菌株改造提高酶产量提供理论依据。