G protein regulation by monoubiquitination

通过单泛素化调节 G 蛋白

• 批准号: 8439313
• 负责人: Henrik G. Dohlman
• 金额: $ 27.64万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8439313
• 关键词: Affect; Animals; Binding; Biochemical; Biological Assay; Categories; Cell Surface Receptors; Cells; Chemicals; Chimera organism; Data; Deubiquitination; Disease; Enzymes; Event; Excision; Family; Feedback; Foundations; Fungal Genome; GTP Binding; GTP-Binding Proteins; GTPase-Activating Proteins; Gene Deletion; Genetic; Genomics; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Health; Hormones; Human; In Vitro; Investigation; Lead; Malignant Neoplasms; Methods; Modification; Mono-S; Monomeric GTP-Binding Proteins; Monoubiquitination; Mutation; Oncogenes; Oncogenic; Phosphorylation; Process; Property; Protein Binding Domain; Protein Subunits; Proteins; Proteomics; Receptor Activation; Refractory; Regulation; Research; Role; Sensory; Signal Transduction; Signaling Protein; Site; Source; Stimulus; System; Testing; Thinking; Time; Ubiquitin; Ubiquitination; Yeasts; base; developmental disease; effective therapy; falls; human disease; in vivo; innovation; mutant; novel strategies; protein degradation; protein protein interaction; protein transport; public health relevance; ras Proteins; receptor; research study; response; tool; trafficking

DESCRIPTION (provided by applicant): Sustained signaling by Ras-family GTPases can lead to cancer as well as developmental disorders. Recent findings reveal that H- and K-Ras are monoubiquitinated and that these modified proteins accumulate in the GTP-bound (activated) state in cells. Our preliminary data reveal that monoubiquitinated Ras is refractory to GTPase activating proteins, but is otherwise fully functional. Based on these findings, we propose that monoubiquitination activates Ras directly, in the absence of a sustained hormone stimulus or oncogenic mutation. Here we propose a comprehensive (in vivo and in vitro) analysis of Ras monoubiquitination in yeast. Aim 1. Functional analysis of monoubiquitinated Ras1. We have shown recently that Ras1, but not Ras2, is monoubiquitinated in yeast. This provides a unique opportunity to compare two functionally similar proteins that are regulated differently. Our hypothesis is that monoubiquitination leads to sustained activation of Ras1. We will identify the structural determinants for selective ubiquitination of Ras1. Using ubiquitination-deficient mutants we will determine how this modification affects Ras signaling in vivo. To establish mechanism, we will determine how monoubiquitination affects the biochemical properties of the protein and its binding partners in vitro. Aim 2. Dynamic regulation of Ras1 monoubiquitination. Our hypothesis is that Ras1 monoubiquitination is a dynamically regulated event. Our preliminary evidence indicates that Ras1 is monoubiquitinated as part of a stimulus-dependent, phosphorylation-dependent feedback mechanism. Using available gene deletions we will determine which enzymes are necessary for the phosphorylation, monoubiquitination and deubiquitination of Ras1. Using purified proteins we will establish which enzymes are sufficient for each modification in vitro. Aim 3 Downstream targets of monoubiquitinated-Ras1. The yeast genome encodes 43 ubiquitin-binding domain (UBD) proteins, which in many cases serve as intracellular "ubiquitin receptors". Our hypothesis is that select UBD proteins interact directly and specifically with monoubiquitinated Ras1, and thereby alter Ras1 trafficking and signaling functions. Using purified proteins and available gene deletion mutants, we will identify the UBDs that target monoubiquitinated Ras1, but not unmodified Ras1 or Ras2. Our approach integrates powerful genomics and proteomics tools, many of which are available only in yeast, to study a process that has clear relevance to human health and disease. If successful, our experiments will reveal (for the first time) proteins responsible for the initiation, addition, removal, and recognition of ubiquitin bound to Ras. Yeast employ a Ras signaling apparatus analogous to that found in humans. Thus a fuller understanding of how Ras signaling is modulated in yeast could eventually lead to fundamentally new approaches to treat Ras-related disease in humans.

描述（由申请人提供）：RAS家庭GTPases的持续信号传导可导致癌症和发育障碍。最近的发现表明，H-和K-RAS是单泛素化的，这些修饰的蛋白质积聚在细胞中的GTP结合（激活）状态中。我们的初步数据表明，单泛素化的RA对GTPase激活蛋白质是难治性的，但否则是完全功能的。基于这些发现，我们建议在没有持续的激素刺激或致癌突变的情况下，单不会直接激活RAS。在这里，我们提出了对酵母中RAS单泛素化的全面（体内和体外）分析。 目标1。单泛素化的RAS1的功能分析。我们最近表明，RAS1（而不是RAS2）在酵母中是单泛素化的。这提供了一个独特的机会，可以比较两个在功能上相似的蛋白质，这些蛋白质的调节方式不同。我们的假设是单泛素化导致RAS1的持续激活。我们将确定RAS1选择性泛素化的结构决定因素。使用泛素化缺陷的突变体我们将确定这种修饰如何影响体内RAS信号传导。为了建立机制，我们将确定单泛素化如何影响蛋白质及其结合伴侣在体外的生化特性。 目标2。RAS1单泛素化的动态调节。我们的假设是RAS1单泛素化是动态调节的事件。我们的初步证据表明，RAS1是刺激依赖性的磷酸化依赖反馈机制的一部分。使用可用的基因缺失，我们将确定哪种酶对于RAS1的磷酸化，单次泛素化和去泛素化是必需的。使用纯化的蛋白质，我们将确定哪些酶在体外的每种修饰都足够。 AIM 3个单液化-RAS1的下游目标。酵母基因组编码43个泛素结合结构域（UBD）蛋白，在许多情况下，它是细胞内“泛素受体”。我们的假设是，选择的UBD蛋白直接与单泛素化的RAS1进行了专门相互作用，从而改变了RAS1运输和信号传导功能。使用纯化的蛋白质和可用的基因缺失突变体，我们将识别靶向单泛素RAS1的UBD，而不是未修饰的RAS1或RAS2。 我们的方法整合了强大的基因组学和蛋白质组学工具，其中许多仅在酵母中可用，以研究与人类健康和疾病显然相关的过程。如果成功的话，我们的实验将揭示（首次）蛋白质，负责与RAS结合的泛素结合的泛素的启动，去除和识别。酵母采用类似于人类的RAS信号传导设备。因此，对酵母中RAS信号的调节方式的更深入了解最终可能导致从根本上产生新的方法来治疗人类中与RAS相关的疾病。