Investigation of mixed ubiquitin chains and chain conformations

泛素化是真核细胞中一种重要的翻译后修饰，调节细胞内广泛的过程。靶分子与泛素的共价修饰不是像磷酸化那样简单的开关信息，而是可以以几种不同的模式发生。该领域的一个关键问题是了解泛素编码是如何产生的，以及它是如何被下游作用因子解码的。泛素密码子的多样性是通过靶蛋白的不同类型修饰实现的：它们可以在特定位点被单个泛素分子修饰（单泛素化），在几个位点被多个泛素分子修饰（多单泛素化）或被各种形式的多聚泛素链修饰。不同链接类型的链与不同的功能相关联。例如，至少四个K48连接的泛素部分的泛素链标记蛋白酶体降解的细胞溶质底物。K11连接的链已被证明在内质网相关蛋白降解（ERAD）和细胞周期控制中发挥重要作用，而K63连接的链可以介导广泛的非蛋白酶体功能，如DNA修复、转录激活、内吞作用或蛋白运输。到目前为止，泛素的研究主要集中在均匀连接的多聚泛素链的研究。然而，越来越多的证据表明，含有一个以上的连接类型的链存在于体内，无论是混合连接链或支链的形式。例如，在细胞中已经检测到K63/M1、K11/K48和K63/K48分支。通过研究ERAD系统中的泛素化过程，我们发现E2酶Ubc 1用K48-连接修饰K63-连接的底物，从而引入K48/K63分支。使用酵母遗传学和生物化学以及生物物理学方法的组合，我们希望进一步表征这种反应，检测体内K48/K63连接，鉴定特异性结合支链的UBD，表征这些相互作用的功能和结构，并研究不同泛素链可以采用的构象。

Ubiquitination is an essential posttranslational modification regulating widespread intracellular processes in eukaryotic cells. Covalent modification of target molecules with ubiquitin is not a simple on-off information like phosphorylation, but can occur in several distinct modes. A key question in the field is to understand how the ubiquitin code is generated and how it is decoded by downstream acting factors. Versatility of the ubiquitin code is achieved by different types of modifications of target proteins: They can either be modified with single ubiquitin molecules at specific sites (mono-ubiquitination), with multiple ubiquitin molecules at several sites (multi mono-ubiquitination) or with various forms of polyubiquitin chains. Chains of different linkage type are associated with different functions. For example, ubiquitin chains of at least four K48-linked ubiquitin moieties mark cytosolic substrates for proteasomal degradation. K11-linked chains have been shown to play important roles in Endoplasmic Reticulum Associated Protein Degradation (ERAD) and cell cycle control, whereas K63-linked chains can mediate widespread non-proteasomal functions such as DNA repair, transcriptional activation, endocytosis or protein trafficking. So far ubiquitin research has focused primarily on the study of homogeneously linked polyubiquitin chains. However there is growing evidence that chains containing more than one linkage type exist in vivo either in form of mixed-linkage-chains or branched chains. For instance K63/M1, K11/K48 and K63/K48 branches have already been detected in cells. By studying ubiquitination processes in the ERAD system we have discovered that the E2 enzyme Ubc1 is decorating K63-linked substrates with K48-linkages thereby introducing K48/K63 branches. Using a combination of yeast genetics and biochemical as well as biophysical methods we want to characterize this reaction further, detect K48/K63 linkages in vivo, identify UBDs specifically binding branched chains, characterize these interactions functionally and structurally and investigate the conformations that different ubiquitin chains can adopt.