The Role Of p97-Chaperone Complexes In Protein Quality Control

描述（由申请人提供）：我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；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压力；内在和外在通过磷酸化网络和泛素介导的信号通路激活适应性反应。我和Melanie Cobb在德克萨斯大学西南分校的研究生工作重点是阐明一个鲜为人知的激酶家族TAOs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损伤反应信号通路的激活很重要。当我加入哈佛大学Wade Harper的实验室，开始研究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低和高含量成像来全局筛选参与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在全球范围内激活导致调节蛋白质降解的途径。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）接触到最先进的筛选技术。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法中的各种方法来解决生物问题。哈佛大学的细胞生物系有大量的资源供博士后相互交流和与教员交流。该部门有一个独特的计划，帮助高级博士后在就业市场上获得技能，成功地导航工作申请过程。我相信这样的环境不仅可以让我提出重要的问题，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。本提案的目标是确定维持细胞中蛋白质质量控制的机制。

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.