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Mechanism and significance of ubiquitin-like protein urmylation in yeast

酵母类泛素蛋白尿酰化的机制及意义

基本信息

批准号：
226230535
负责人：
Professor Dr. Raffael Schaffrath
金额：
--
依托单位：
Fachgebiet Mikrobiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/226230535?language=en
关键词：
Mechanism significance ubiquitin like protein

项目摘要

Ubiquitin-related modifier 1 (Urm1) from budding yeast is a ubiquitin family (UbF) member with protein and tRNA modification functions typical of eukaryal UbF modifiers and prokaryal sulphur carriers, respectively. Despite its well documented role in tRNA modification/thiolation, only one protein (Ahp1) conjugated to Urm1 is known to date from yeast stressing that both the significance of urmylation and Urm1 target identity are open issues in Saccharomyces cerevisiae. Nonetheless, based on the recent isolation of various human URM1 targets, assigning biological functions to protein urmylation seems feasible. Therefore, we propose two complementary strategies for studying Urm1 pathways in yeast. In the first, bioinformatics will identify selected yeast homologs of human targets for direct urmylation and cross-complementation assays. This has the potential to identify bona fide Urm1 targets and study conservation within Urm1 modification pathways among eukaryotes. The second uses a TAP-tagged bait of Urm1 for proteome-wide Urm1 target identification in yeast by mass spectrometry. To validate urmylation, candidates from each approach will be assessed by independent Urm1 conjugation assays and bona fide Urm1 interactions are correlated with phenotypic profiles obtained from strains mutated in the appropriate Urm1 target genes. The latter holds promise to ask how urmylation may affect target protein function and importantly, whether protein urmylation and tRNA thiolation represent related or independent branches of Urm1 modification pathways. In sum, by combining molecular, biochemical and bioinformatical methods, the proposal is likely to provide new insights into the biology and significance of the eukaryal Urm1 modification pathway.

来自芽殖酵母的Ubiquitin-related modifiers 1（Urm 1）是泛素家族（UbF）成员，具有真核UbF修饰剂和原核硫载体的蛋白质和tRNA修饰功能。尽管其在tRNA修饰/硫醇化中的作用得到了充分的证明，但迄今为止，已知酵母中只有一种与Urm 1缀合的蛋白质（Ahp 1），这表明urmylation的意义和Urm 1靶向身份都是酿酒酵母中的未决问题。尽管如此，基于最近分离的各种人类URM 1目标，分配生物功能的蛋白尿嘧啶化似乎是可行的。因此，我们提出了两个互补的策略，研究Urm 1途径在酵母。在第一，生物信息学将确定选定的酵母同源物的人类目标的直接urmylation和交叉互补测定。这有可能确定真正的Urm 1目标和研究真核生物之间的Urm 1修饰途径内的保护。第二种方法使用TAP标记的Urm 1诱饵，通过质谱法在酵母中进行蛋白质组范围的Urm 1目标识别。为了验证urmylation，将通过独立的Urm 1结合试验评估每种方法的候选物，并将真正的Urm 1相互作用与从适当Urm 1靶基因突变的菌株获得的表型谱相关联。后者有希望问如何urmylation可能会影响靶蛋白的功能，重要的是，蛋白urmylation和tRNA巯基化是否代表相关或独立的分支Urm 1修饰途径。综上所述，通过结合分子生物学、生物化学和生物信息学的方法，该方案可能为真核生物Urm 1修饰途径的生物学和意义提供新的见解。