Investigation of mixed ubiquitin chains and chain conformations

混合泛素链和链构象的研究

• 批准号: 394619424
• 负责人: Professor Dr. Volker Dötsch
• 金额: --
• 依托单位: Forschungsgruppe Intrazelluläre Proteolyse
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/394619424?language=en
• 关键词: Investigation; mixed; ubiquitin; chains; chain; Investigation; mixed; ubiquitin; chains; chain

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.

泛素化是一种基本的翻译后修饰，可调节真核细胞中广泛的细胞内过程。靶分子与泛素的共价修饰不是诸如磷酸化之类的简单开关信息，而是以几种不同的模式发生。该领域中的一个关键问题是了解如何生成泛素代码以及如何通过下游作用因素解码。泛素代码的多功能性是通过靶蛋白的不同类型的修饰来实现的：可以在特定位点（单次泛素化）在多种位点（多种单个单个单位）（多单单求的）（多种单单求）在特定位点（单次泛素分子）在特定位点（单次泛素分子）进行修饰。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志着蛋白酶体降解的胞质底物。已经显示，K11连接的链在内质网中起重要作用，在内质网和细胞周期控制中起着重要作用，而K63连接的链可以介导广泛的非骨膜函数，例如DNA，诸如DNA修复，转录激活，内吞和吞噬蛋白质。到目前为止，泛素研究主要集中在均质连接的多泛素链的研究上。然而，越来越多的证据表明，在体内以混合链链或分支链的形式存在含有多种连锁的链条。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UBC1正在装饰具有K48-链接的K63连接底物，从而引入了K48/K63分支。使用酵母遗传学和生化方法以及生物物理方法的结合，我们希望进一步表征该反应，检测体内的K48/K63链接，识别特异性结合的分支链，在功能和结构上表征这些相互作用，并在结构上和结构上表征这些相互作用，并研究不同的构象，而不同的ubiquitin链可以采用。