The Role Of p97-Chaperone Complexes In Protein Quality Control

p97-伴侣复合物在蛋白质质量控制中的作用

基本信息

批准号：
9140089
负责人：
Malavika Raman
金额：
$ 18.02万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-15 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9140089
关键词：
ATP phosphohydrolase Aneuploid Cells Aneuploidy Binding Biological CDT1 Gene Cell Culture Techniques Cell Cycle Proteins Cell Proliferation Cells Cellular Stress Cellular biology Chemistry Chromatin Chromosomes Client Collaborations Complex Cytosol DNA Damage DNA biosynthesis Degradation Pathway Destinations Disease Docking Endoplasmic Reticulum Environment Equilibrium Event Faculty Family Functional disorder Genes Genetic Screening Goals Health Homeostasis Human Genome Image In Vitro Institutes Intervention Job Application Lead Location Malignant Neoplasms Mass Spectrum Analysis Mediating Methods Modeling Molecular Chaperones Normal Cell Pathway interactions Peptides Phosphorylation Phosphotransferases Postdoctoral Fellow Prions Process Production Protein Biosynthesis Protein Region Proteins Proteome Proteomics Quality Control Recruitment Activity Regulation Replication Licensing Reporter Research Resort Resources Ribosomes Role Signal Pathway Signal Transduction Small Interfering RNA Stable Isotope Labeling Stress System Technology Therapeutic Intervention Toxic effect Translating Triage Ubiquitin Ubiquitination Up-Regulation Veins Vision Work addiction cancer cell cohort coping hands-on learning in vitro activity in vivo interest job market knock-down member multicatalytic endopeptidase complex neoplastic cell non-Native novel oncogene addiction p97 ATPase polypeptide preprolactin programs protein degradation protein expression protein misfolding reconstitution research facility research study response screening skills small molecule inhibitor targeted treatment ubiquitin-protein ligase unfoldase

项目摘要

DESCRIPTION (provided by applicant): The long-term vision for my research is to determine how the ubiquitin proteasome system (UPS) can be targeted for therapy in diseases such as cancer; the short-term goals of this proposal are to understand the fundamentals of how the UPS regulates protein quality control. My past research has focused on how cellular stress; both intrinsic and extrinsic activate adaptive responses via activation of phosphorylation networks and ubiquitin-mediated signaling pathways. My graduate work with Melanie Cobb at UT Southwestern focused on elucidating signaling pathways involving a poorly understood family of kinases known as TAOs. My work led to the important finding that these kinases were important for the activation of DNA damage responsive signaling pathways. I continued in this vein when I joined Wade Harper's lab at Harvard and initially studied how DNA damage led to the ubiquitin-mediated degradation of a key cell cycle protein known CDT1. Importantly, I developed a screening platform that allowed me to globally screen for genes involved in CDT1 degradation using siRNA mediated knockdown of all the genes in the human genome and high-content imaging. I am currently interested in applying quantitative mass- spectrometry to understand how cellular stress globally activates pathways leading to regulated protein degradation. In this regard the environment at Harvard is key, I have already been exposed to state of the art screening technologies at the Institute of Chemistry and Cell Biology (ICCB) at Harvard. The Harper lab's long- standing collaboration with Steve Gygi's lab at Harvard has enabled members of the Harper lab to learn and apply various methods in mass spectrometry to biological problems. The department of Cell Biology at Harvard has numerous resources for post-doctoral fellows to interact with each other and with faculty. The department has a unique program that helps senior post-docs on the job market with acquiring the skills to successfully navigate the job application process. I believe that such an environment allows me not only to ask important questions, but also provides the facilities and research expertise to help me answer them. The goal of this proposal is to identify the mechanisms that maintain protein quality control in cells. As proteins are translated off the ribosome, chaperone systems are in place to bind the polypeptide and assist in their folding and targeting. This is particularly important for proteins with hydrophobic regions that are normally buried in the folded state, but are exposed during protein synthesis. If these regions are not appropriately shielded from the cytosol, they will aggregate and lead to toxicity. A growing body of research indicates that aneuploid tumor cells express the proteins encoded in their extra chromosomes. Expression of these proteins may be potentially harmful to cells because it imbalances the normal repertoire of cellular proteins and overburdens chaperone systems. Cancer cells are thought to overcome this adversity by up-regulating chaperones for assisted folding. This has been termed a form of 'non-oncogene' addiction. In general, during cases of proteomic stress, the excess of newly synthesized proteins that fail to fold is ubiquitinated and destroyed by the proteasome. Recently a complex of chaperones has been identified that associate with the translating ribosome and bind to hydrophobic regions of proteins when they are released into the cytosol. This system, nucleated on the BAG6 chaperone aids in the insertion of these proteins into endoplasmic reticulum (ER) where they will be processed for their final destination. It has been shown that when hydrophobic domain containing proteins fail to translocate into the ER, the BAG6 chaperone complex aids in their ubiquitination and degradation. However, little is known about the proteins that ubiquitinate and facilitate degradation of BAG6 client proteins. Importantly, most of the studies have been performed with a handful of reporter substrates, so it is unclear what the full cohort of BAG6 substrates in cells are. Through a proteomic screen, we have identified two new components of this pathway that associate with BAG6. The studies proposed here will attempt to identify the role of these complexes in the degradation of newly synthesized proteins, reconstitute their activity in vitro and identify their relevant targets in cancer cells. Overall tis proposal will elaborate on the mechanisms that maintain proteostais and identify new avenues for intervention in cancer and other aggregation prone diseases.

描述（由申请人提供）：我的研究的长期愿景是确定如何将泛素蛋白酶体系统（UPS）靶向治疗癌症等疾病；该提案的短期目标是了解 UPS 如何调节蛋白质质量控制的基本原理。我过去的研究重点是细胞压力如何；内在和外在都通过激活磷酸化网络和泛素介导的信号通路来激活适应性反应。我与 UT Southwestern 的 Melanie Cobb 的研究生工作重点是阐明涉及人们知之甚少的激酶家族（TAO）的信号通路。我的工作得出了一个重要发现，即这些激酶对于激活 DNA 损伤响应信号通路非常重要。当我加入哈佛大学 Wade Harper 的实验室时，我继续沿着这个思路，最初研究 DNA 损伤如何导致泛素介导的关键细胞周期蛋白 CDT1 的降解。重要的是，我开发了一个筛选平台，使我能够使用 siRNA 介导的人类基因组中所有基因的敲除和高内涵成像来全面筛选参与 CDT1 降解的基因。我目前感兴趣的是应用定量质谱法来了解细胞应激如何全局激活导致受调节的蛋白质降解的途径。在这方面，哈佛大学的环境是关键，我已经在哈佛大学化学与细胞生物学研究所（ICCB）接触过最先进的筛选技术。哈珀实验室与哈佛大学 Steve Gygi 实验室的长期合作使哈珀实验室的成员能够学习和应用质谱中的各种方法来解决生物问题。哈佛大学细胞生物学系拥有大量资源，供博士后研究员相互交流以及与教师互动。该部门有一个独特的计划，可以帮助就业市场上的高级博士后获得成功完成工作申请流程的技能。我相信这样的环境不仅让我能够提出重要的问题，而且还提供了帮助我回答这些问题的设施和研究专业知识。该提案的目标是确定维持细胞中蛋白质质量控制的机制。当蛋白质从核糖体翻译出来时，伴侣系统就位以结合多肽并协助其折叠和靶向。这对于具有疏水区域的蛋白质尤其重要，这些区域通常隐藏在折叠状态，但在蛋白质合成过程中暴露出来。如果这些区域没有适当地与细胞质隔离，它们就会聚集并导致毒性。越来越多的研究表明，非整倍体肿瘤细胞表达其额外染色体中编码的蛋白质。这些蛋白质的表达可能对细胞有害，因为它会破坏细胞蛋白质的正常功能并加重伴侣系统的负担。人们认为癌细胞通过上调辅助折叠的分子伴侣来克服这种逆境。这被称为“非癌基因”成瘾的一种形式。一般来说，在蛋白质组应激的情况下，无法折叠的过量新合成蛋白质被泛素化并被蛋白酶体破坏。最近，已鉴定出一种分子伴侣复合物，其与翻译核糖体相关，并在蛋白质释放到胞质溶胶中时与蛋白质的疏水区域结合。该系统以 BAG6 分子伴侣为核，有助于将这些蛋白质插入内质网 (ER)，在那里它们将被加工至最终目的地。研究表明，当含有疏水结构域的蛋白质无法转位至内质网时，BAG6 分子伴侣复合物会帮助其泛素化和降解。然而，人们对泛素化并促进 BAG6 客户蛋白降解的蛋白知之甚少。重要的是，大多数研究都是使用少数报告底物进行的，因此尚不清楚细胞中完整的 BAG6 底物群是什么。通过蛋白质组筛选，我们确定了该通路中与 BAG6 相关的两个新成分。这里提出的研究将试图确定这些复合物在新合成蛋白质降解中的作用，在体外重建它们的活性，并确定它们在癌细胞中的相关靶标。总体而言，该提案将详细阐述维持蛋白质稳定的机制，并确定干预癌症和其他易聚集疾病的新途径。