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

• 批准号: 9321274
• 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
• 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/9321274
• 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; Destinations; Disease; Docking; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Environment; Equilibrium; Event; Faculty; Family; Functional disorder; Genes; Genetic Screening; Goals; Homeostasis; Human Genome; Hydrophobicity; Image; In Vitro; Institutes; Intervention; Job Application; Lead; Learning; Location; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Modeling; Molecular Chaperones; Mutation Analysis; 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; Ribosomal Proteins; 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; cancer cell; cohort; experimental study; in vitro activity; in vivo; interest; job market; knock-down; member; misfolded protein; multicatalytic endopeptidase complex; neoplastic cell; non-Native; novel; p97 ATPase; polypeptide; premature; preprolactin; programs; protein degradation; protein expression; protein reconstitution; proteostasis; proteotoxicity; public health relevance; reconstitution; response; screening; skills; small molecule inhibitor; targeted treatment; ubiquitin-protein ligase; unfoldase

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）用于癌症等疾病的治疗;本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。我过去的研究主要集中在细胞应激如何通过激活磷酸化网络和泛素介导的信号通路来激活内源性和外源性适应性反应。我与UT西南大学的Melanie Cobb的研究生工作集中在阐明涉及一个鲜为人知的激酶家族（TAO）的信号通路。我的工作导致了一个重要的发现，即这些激酶对DNA损伤响应信号通路的激活很重要。当我加入哈佛的韦德哈珀实验室，并开始研究DNA损伤如何导致泛素介导的关键细胞周期蛋白CDT 1的降解时，我继续了这一思路。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含量成像来全球筛选参与CDT 1降解的基因。我目前感兴趣的是应用定量质谱来了解细胞应激如何在全球范围内激活导致蛋白质降解的途径。在这方面，哈佛的环境是关键，我已经在哈佛的化学和细胞生物学研究所（ICCB）接触到了最先进的筛选技术。哈珀实验室与哈佛大学史蒂夫·吉吉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分析中的各种方法来解决生物问题。哈佛的细胞生物学系为博士后研究员提供了许多资源，使他们能够相互交流并与教师交流。该部门有一个独特的计划，帮助就业市场上的高级博士后获得成功浏览工作申请过程的技能。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。 当蛋白质被翻译离开核糖体时，分子伴侣系统就在适当的位置结合多肽并帮助它们折叠和靶向。这对于具有疏水区域的蛋白质特别重要，这些疏水区域通常以折叠状态隐藏，但在蛋白质合成期间暴露。如果这些区域没有适当地与细胞质屏蔽，它们将聚集并导致毒性。越来越多的研究表明，非整倍体肿瘤细胞表达编码在其额外染色体中的蛋白质。这些蛋白质的表达可能对细胞有害，因为它使细胞蛋白质的正常库失衡并使伴侣系统负担过重。癌细胞被认为通过上调分子伴侣来辅助折叠来克服这种逆境。这被称为“非癌基因”成瘾。一般来说，在蛋白质组应激的情况下，过量的新合成的不能折叠的蛋白质被泛素化并被蛋白酶体破坏。最近，已经鉴定出与翻译核糖体缔合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的伴侣蛋白复合物。该系统在BAG 6分子伴侣上成核，有助于将这些蛋白质插入内质网（ER），在内质网中它们将被加工成其最终目的地。已经表明，当含有疏水结构域的蛋白质不能易位到ER中时，BAG 6伴侣复合物有助于它们的泛素化和降解。然而，关于BAG 6客户蛋白的泛素化和促进降解的蛋白质知之甚少。重要的是，大多数研究都是用少数报告底物进行的，因此尚不清楚细胞中BAG 6底物的完整队列是什么。通过蛋白质组学筛选，我们已经确定了与BAG 6相关的该途径的两个新组分。本文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，在体外重建其活性并确定其在癌细胞中的相关靶点。总的来说，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聚集倾向性疾病的新途径。