The Role Of p97-Chaperone Complexes In Protein Quality Control

描述(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)用于癌症等疾病的治疗；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原理。我过去的研究集中在细胞应激；内在和外在如何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。我和梅勒妮·科布在德克萨斯大学西南分校的研究生工作重点是阐明信号通路，涉及一个鲜为人知的被称为Taos的激酶家族。我的工作导致了一个重要的发现，即这些激酶对于激活DNA损伤反应信号通路是重要的。当我加入哈佛大学韦德·哈珀的实验室时，我延续了这种思路，最初研究了DNA损伤如何导致一种名为CDT1的关键细胞周期蛋白的泛素介导的降解。重要的是，我开发了一个筛选平台，使我能够通过siRNA介导的人类基因组中所有基因的敲除和高含量成像，在全球范围内筛选与CDT1降解有关的基因。我目前感兴趣的是应用定量质谱学来了解细胞应激如何在全球范围内激活导致受调控的蛋白质降解的途径。在这方面，哈佛的环境是关键，我已经接触到哈佛化学和细胞生物学研究所(ICCB)最先进的筛选技术。哈珀实验室与哈佛大学史蒂夫·吉吉的实验室长期合作，使哈珀实验室的成员能够学习和应用质谱学中的各种方法来解决生物问题。哈佛大学细胞生物学系有许多资源，供博士后研究员相互交流和与教师互动。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通过工作申请过程的技能。我相信，这样的环境不仅让我可以提出重要的问题，还提供了帮助我回答这些问题的设施和研究专业知识。这项提议的目标是确定维持细胞内蛋白质质量控制的机制。 当蛋白质被翻译出核糖体时，伴侣系统就会到位，以结合多肽并帮助其折叠和靶向。这对于具有疏水区域的蛋白质来说尤其重要，这些蛋白质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屏蔽细胞质，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在其额外染色体中编码的蛋白质。这些蛋白质的表达可能会对细胞造成潜在的伤害，因为它会扰乱正常的细胞蛋白质谱，并使伴侣系统负担过重。癌细胞被认为可以通过上调辅助折叠的伴侣蛋白来克服这一不利因素。这被称为“非癌基因”上瘾的一种形式。一般来说，在蛋白质组应激的情况下，新合成的不能折叠的多余蛋白质被泛素化，并被蛋白酶体破坏。最近，人们发现了一种与翻译核糖体相关的伴侣复合体，当蛋白质释放到胞浆中时，它会与蛋白质的疏水性区域结合。这个位于BAG6分子伴侣上的系统有助于将这些蛋白质插入内质网(ER)，在那里它们将被加工成最终的目的地。研究表明，当含有疏水结构域的蛋白质不能转移到内质网时，BAG6伴侣复合体有助于它们的泛素化和降解。然而，人们对泛素化和促进BAG6客户蛋白降解的蛋白质知之甚少。重要的是，大多数研究都是在少数报告底物的情况下进行的，因此还不清楚细胞中BAG6底物的完整队列是什么。通过蛋白质组学筛选，我们已经确定了该途径中与BAG6相关的两个新成分。本文提出的研究将试图确定这些复合体在降解新合成的蛋白质中的作用，在体外重建它们的活性，并确定它们在癌细胞中的相关靶点。总的TIS提案将详细阐述维持蛋白质组分的机制，并确定干预癌症和其他容易聚集的疾病的新途径。

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.