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）：我的研究的长期视力是确定泛素蛋白酶体系统（UPS）如何针对癌症等疾病的治疗；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面。我过去的研究集中在细胞压力上。固有和外在的通过激活磷酸化网络和泛素介导的信号传导途径激活适应性反应。我在UT西南部与梅兰妮·科布（Melanie Cobb）的研究生工作着重于阐明信号通路，涉及一个知识熟悉的激酶家族，称为陶斯（Taos）。我的工作导致了一个重要发现，即这些激酶对于激活DNA损伤响应信号通路很重要。当我加入哈佛大学的韦德·哈珀（Wade Harper）实验室时，我继续使用这种脉络，并最初研究了DNA损伤如何导致泛素介导的关键细胞周期蛋白已知CDT1的降解。重要的是，我开发了一个筛选平台，该平台使我能够使用siRNA介导的人类基因组和高含量成像中所有基因的siRNA介导的敲低CDT1降解的基因进行筛选。我目前有兴趣应用定量质谱法以了解细胞应力如何激活导致蛋白质降解的途径。在这方面，哈佛大学的环境是关键，我已经接触到哈佛化学和细胞生物学研究所（ICCB）的最先进的筛查技术。哈珀实验室与哈佛大学史蒂夫·吉吉（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，以解决生物学问题。哈佛大学的细胞生物学系有许多资源用于博士后研究员，可以与彼此互动并与教师互动。该部门拥有一个独特的计划，可以帮助获得工作市场的高级职位，以获取成功浏览工作申请流程的技能。我相信这样的环境不仅可以提出重要问题，还可以提供设施和研究专业知识来帮助我回答它们。该建议的目的是确定维持细胞蛋白质质量控制的机制。随着蛋白质从核糖体转换，伴侣系统就可以结合多肽并有助于其折叠和靶向。这对于通常埋在折叠状态但在蛋白质合成过程中暴露的疏水区域的蛋白质尤为重要。如果这些区域没有适当地避免侵害细胞质，它们将汇总并导致毒性。越来越多的研究表明，非整倍型肿瘤细胞表达在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，因为它会失去细胞蛋白和覆盖伴侣系统的正常曲目。人们认为癌细胞通过上调伴侣以辅助折叠来克服这种逆境。这被称为“非癌基因”成瘾形式。通常，在蛋白质组学胁迫的情况下，未能折叠的新合成蛋白的过量被蛋白酶体泛素化和破坏。最近，已经确定了伴侣的复合体，该复合体与翻译核糖体相关，并与蛋白质的疏水区结合，当它们释放到胞质溶胶中时。该系统将这些蛋白质插入内质网中（ER）插入BAG6伴侣辅助辅助物（ER），并将它们用于最终目的地。已经表明，当含有蛋白质的疏水结构域未能转移到ER中时，Bag6伴侣复合物有助于其泛素化和降解。但是，对于泛素化和促进BAG6客户蛋白降解的蛋白质知之甚少。重要的是，大多数研究都是用少数记者底物进行的，因此尚不清楚细胞中的BAG6底物的完整队列。通过蛋白质组学屏幕，我们确定了与Bag6相关的该途径的两个新组件。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，在体外重建其活性，并确定其在癌细胞中的相关靶标。总体提案将详细阐述维持蛋白质的机制，并确定干预癌症和其他容易发生疾病的新途径。