Dissecting the Function and Regulation of Stress-Induced SUMOylation

剖析压力诱导的 SUMOylation 的功能和调节

• 批准号: 8398134
• 负责人: Robert Charles Augustine
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398134
• 关键词: Acute; Affect; Angiosperms; Bacteria; Binding; Biological Assay; Bypass; Cataloging; Catalogs; Cellular Stress Response; ChIP-seq; Chromatin; Consensus; Defect; Dissection; Enzymes; Eukaryota; Eukaryotic Cell; Foundations; Future; Gene Expression; Gene Proteins; Genes; Genome; Genotoxic Stress; Goals; Heat Stress Disorders; Herpesvirus 1; Host Defense; Human Adenoviruses; In Vitro; Infection; Lead; Ligase; Link; Listeria monocytogenes; Malignant neoplasm of liver; Mammalian Cell; Mediating; Messenger RNA; Metabolism; Methods; Microbe; Modification; Mouse-ear Cress; Organism; Output; Pathogenesis; Pathway interactions; Plants; Proteins; Proteomics; RNA; RNA Splicing; RNA-Binding Proteins; Reaction; Regulation; Research Project Grants; Role; Streptococcus pneumoniae; Stress; Sumoylation Pathway; Testing; Transcription Coactivator; Transcriptional Regulation; Ubiquitin; Ubiquitination; Viral; Virus; Work; base; biological adaptation to stress; chromatin modification; chronic liver disease; coping; defense response; genome-wide; human disease; in vivo; influenzavirus; insight; loss of function; mutant; next generation; novel; pathogen; pathogenic bacteria; plant fungi; research study; response; stress tolerance; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): In eukaryotes, stress rapidly triggers the conjugation of small ubiquitin-related modifier (SUMO) to intracellular target proteins to alter their activity and mount a defense response. Defects in stress-induced SUMOylation (SIS) are associated with a number of human diseases including acute and chronic liver disease and cancer induced by genotoxic stress. In addition, several pathogenic bacteria (Listeria monocytogenes, Streptococcus pneumoniae) and viruses (human adenovirus, influenza virus) alter SIS, and this is important for bypassing defenses and promoting infection. Like metazoans, several plant pathogens hijack the SUMOylation pathway to render host defense ineffectual, suggesting that this pathway serves a conserved role in defense against biotic attack. Thus, it is essential that we determine precisely how SUMOylation triggers changes that minimize stress damage, inhibit pathogenesis, and promote survival. In particular, a long-term objective of this study is to determine how the SIS response mediates downstream effects that lead to stress protection. The flowering plant Arabidopsis thaliana is well suited for understanding the role of SUMOylation during stress. It has characterized mutants affecting the SIZ1 ligase that drives SIS, which are hypersensitive to many stresses and fail to elicit the SIS response; thereby permitting the dissection of SUMOylation during stress. In addition, substantial overlap between SUMO targets in plants and metazoans suggests a conserved response that converges on factors important for transcriptional regulation, RNA metabolism, and chromatin modification, indicating that the SIS response universally affects gene expression and chromatin accessibility. To identify the transcriptional and chromatin alterations associated with the SIS response, comparisons of wild type and siz1 mutant plants will be performed before and after heat stress. Specifically, next-generation sequencing will be used to track changes in SUMO1/2 binding occupancy along chromatin (ChIP-Seq) and the alterations to mRNA abundance/splicing (RNA-Seq). Changes occurring in wild type plants that are absent in siz1 mutants will define how SUMOylation rewires the genome in response to stress and, in doing so, has the potential to identify novel genes involved in stress tolerance. Some SUMO targets are also modified with ubiquitin specifically during stress with their dynamics suggesting that they are targeted for degradation. A second focus of this proposal aims to identify dual- modified targets using a novel proteomics approach. These targets will assist in identifying candidate SUMO- targeted ubiquitin ligases that catalyze this reaction. Loss-of-function analyses before and after heat stress will establish the importance and function of this dual modification during adverse conditions. Collectively, these experiments aim to provide a comprehensive understanding of the outputs and regulation of the SIS response. Broadly, this work will provide a mechanistic basis for understanding how organisms can rapidly respond and defend themselves from environmental stress and pathogen invasion. PUBLIC HEALTH RELEVANCE: In response to pathogen invasion and abiotic stress, eukaryotic cells rapidly attach small ubiquitin-like modifier (SUMO) to an array of target proteins to alter their activity and protect against and minimize the damage caused by these stresses. Defects in stress-induced SUMOylation have been implicated in human diseases, where targeted disruption of the SUMOylation pathway by Herpes Simplex Virus Type 1, Streptococcus pneumoniae, Listeria monocytogenes, and other bacterial and viral invaders is linked with pathogenesis. Therefore, determining how stress-induced SUMOylation is regulated and the means by which this modification serves a protective role under stress conditions will be fundamentally important to fully understand a variety of normal cellular stress responses as well as human disease states.

描述(申请人提供)：在真核生物中，应激可迅速触发小泛素相关修饰物(SUMO)与细胞内靶蛋白的结合，从而改变其活性 并做出防御反应。应激诱导的糖基化缺陷(SIS)与许多人类疾病有关，包括急、慢性肝病和由基因毒性应激引起的癌症。此外，几种致病细菌(李斯特菌、肺炎链球菌)和病毒(人腺病毒、流感病毒)改变SIS，这对于绕过防御和促进感染是重要的。像后生动物一样，几种植物病原体劫持了SUMO化途径，使宿主的防御无效，这表明这一途径在防御生物攻击中起着保守的作用。因此，我们必须准确地确定SUMO化如何触发变化，从而将应激损伤降至最低，抑制发病机制，并促进生存。特别是，这项研究的长期目标是确定SIS的反应如何调节导致压力保护的下游效应。开花植物拟南芥非常适合于理解SUMO化在逆境中的作用。它的特征是影响驱动SIS的SIZ1连接酶的突变体，这些突变体对许多应激反应高度敏感，无法引发SIS反应，从而允许在应激过程中分解SUMO化。此外，植物和后生动物中的相扑靶标之间的大量重叠表明，一种保守的反应聚集在转录调控、RNA代谢和染色质修饰的重要因子上，表明SIS反应普遍影响基因表达和染色质的可及性。为了确定与SIS反应相关的转录和染色质变化，将对野生型和siz1突变植株在热胁迫前后进行比较。具体地说，下一代测序将用于跟踪SUMO1/2结合占有率沿染色质的变化(ChIP-Seq)和mRNA丰度/剪接的变化(RNA-Seq)。在野生型植物中发生的变化，这些变化在siz1突变体中缺失，将定义SUMO化如何重新连接基因组以响应压力，并在这样做的过程中，有可能发现参与逆境耐受的新基因。一些相扑靶标在应激期间也会被泛素修饰，它们的动态变化表明它们是降解的靶标。这项提案的第二个重点是使用一种新的蛋白质组学方法来识别双重修饰靶点。这些靶标将有助于确定催化这一反应的相扑靶标泛素连接酶候选。热应激前后的功能丧失分析将确定这种双重修饰在不利条件下的重要性和作用。总而言之，这些实验旨在提供对SIS反应的输出和调节的全面了解。广泛地说，这项工作将为理解生物体如何快速反应和防御环境压力和病原体入侵提供机制基础。 公共卫生相关性：为了应对病原体入侵和非生物应激，真核细胞迅速将小泛素样修饰物(SUMO)附着到一系列靶蛋白上 以改变他们的活动，防止和尽量减少这些压力造成的损害。应激诱导的SUMO化缺陷与人类疾病有关，其中单纯疱疹病毒1型、肺炎链球菌、单核细胞增生性李斯特菌和其他细菌和病毒侵入者对SUMO化途径的靶向性干扰与发病有关。因此，确定应激诱导的SUMO化是如何调节的，以及这种修饰在应激条件下发挥保护作用的方式，对于全面理解各种正常的细胞应激反应以及人类的疾病状态将是至关重要的。