High throughput structure/function analysis of SUMO modification

描述(由申请人提供)：项目摘要SUMOS(小泛素相关修饰物)是翻译后与其他蛋白质连接的蛋白质，从而调节对正常细胞生长、分化和生存至关重要的一系列重要功能。值得注意的是，苏莫化与多种人类疾病的发展和进展直接相关，包括神经退行性变、中风、心脏病、唇腭裂和癌症。在过去的十年里，在阐明SUMOS与靶蛋白结合的分子机制以及鉴定SUMOS所调控的细胞过程方面取得了巨大的进展。然而，尽管有这些见解，关于苏莫化实际上如何在分子水平上影响修饰的蛋白质，以及这些影响如何转化为对细胞功能的控制的基本问题仍有待解决。在许多情况下，和甲基化对靶蛋白的影响仍不清楚。此外，苏莫化如何能够在与不同的蛋白质结合时产生独特的命运也鲜为人知。我们将使用发芽酵母酿酒酵母作为模式生物来研究苏莫化的基本功能。这项研究的目标之一是开发高分辨率相扑结构/功能图。这一目标将通过合成一个全面的酵母相扑突变体文库并使用各种旨在探索相扑结构/功能关系的生化和遗传分析来鉴定这些突变体来实现。有了这个文库，我们将检验这一假设，即相扑代表一个多功能和可变的信号，具有不同的表面残基，影响与不同调控因子和下游相扑结合蛋白的相互作用。我们还将测试这一假设，即苏莫化以一种类似于伴侣的能力来保护蛋白质免受细胞毒细胞压力的影响而造成的损伤和聚集。

DESCRIPTION (provided by applicant): PROJECT SUMMARY SUMOs (small ubiquitin-related modifiers) are proteins that are posttranslationally conjugated to other proteins, thereby regulating a wide range of essential functions critical for normal cell growth, differentiation and survival. Notably, sumoylation has been directly linked to the development and progression of a variety of human diseases, including neurodegeneration, stroke, heart disease, cleft lip/palate and cancer. During the past ten years enormous progress has been made in elucidating the molecular mechanisms involved in conjugating SUMOs to target proteins and in identifying the cellular processes regulated by sumoylation. Despite these insights, however, fundamental questions about how sumoylation actually affects modified proteins at the molecular level, and how these effects are translated into control of cell function, remain to be addressed. In many cases, the effects of sumoylation on target proteins remain unknown. In addition, how sumoylation is able to elicit unique fates upon conjugation to different proteins is also poorly understood. We will use the budding yeast, S. cerevisiae, as a model organism for studying the essential functions of sumoylation. A goal of the proposed studies is develop a high-resolution structure/function map of SUMO. This goal will be achieved through the synthesis of a comprehensive library of yeast SUMO mutants and characterization of these mutants using a variety of assays designed to probe SUMO structure/function relationships both biochemically and genetically. With this library, we will test the hypothesis that SUMO represents a versatile and variable signal with distinct surface residues affecting interactions with distinct regulatory factors and downstream SUMO-binding proteins. We will also test the hypothesis that sumoylation functions in a chaperone-like capacity to protect proteins from damage and aggregation caused by the effects of cytotoxic cell stresses.