New Ubiquitin-Proteasome System Components that Protect against Proteotoxicity

描述（由申请人提供）：细胞生物学的一个核心问题是细胞如何破坏自己的蛋白质。蛋白质错误折叠是对细胞的普遍威胁，有多种原因，包括热、翻译错误、DNA损伤、重金属和类金属、遗传突变、氧化和衰老。蛋白质错误折叠也与大多数神经退行性疾病和某些癌症类型有关。泛素-蛋白酶体系统，凭借其破坏蛋白质的能力，对蛋白质错误折叠的威胁作出反应，有时被称为蛋白质毒性。我们已经确定了这种应激反应途径的一个新分支，它专门保护细胞免受三价类金属（如砷）诱导的错误折叠蛋白质的影响，而不是其他原因导致的蛋白质错误折叠。这一反应途径的关键介质是Cuz1，以及相关但在很大程度上未被表征的蛋白Tmc1。我们发现Cuz1作为一种新的锌依赖性泛素结合蛋白，与蛋白酶体和多功能伴侣蛋白Cdc48/p97相互作用。这些数据表明Cuz1通过识别错误折叠的蛋白质并将其传递给蛋白酶体进行破坏来保护细胞免受三价类金属的侵害。我工作的长期目标是更好地理解细胞内蛋白质降解是如何发生的。在这个应用中，我结合了生物化学、遗传学、细胞生物学、蛋白质组学和结构的方法来了解Cuz1和Tmc1介导的应激反应途径。首先，我将描述Cuz1的泛素结合功能。我的数据表明在Cuz1中有一个潜在的新的进化保守的泛素识别基序。如果得到验证，Cuz1可能代表一类新的泛素结合蛋白的创始成员。其次，我将利用蛋白质组学方法鉴定Cuz1的特定底物。由于Cuz1在保护细胞免受金属诱导的蛋白质毒性方面具有显著的特异性，这些研究可能为蛋白质降解中最重要的问题之一提供见解，该问题与特异性如何产生和维持有关。第三，我们将首次对Tmc1进行重要的表征，Tmc1是一种与Cuz1序列和表型相似的锌指蛋白。我将确定Cuz1和Tmc1如何协调它们的保护性应激反应。总之，这些目标将提供对细胞生物学基本方面的见解，调节蛋白质降解，这样做将告知与错误折叠蛋白质相关的许多疾病。

DESCRIPTION (provided by applicant): A central question in cell biology concerns how cells destroy their own proteins. Protein misfolding represents a universal threat to cells, and has numerous causes including heat, errors in translation, DNA damage, heavy metals and metalloids, inherited mutation, oxidation, and aging. Protein misfolding is also associated with most neurodegenerative disease and some cancer types. The ubiquitin-proteasome system, by virtue of its ability to destroy proteins, responds to the threat of protein misfolding, sometimes termed proteotoxicity. We have identified a novel arm of this stress response pathway that specifically protects cells from misfolded proteins induced by trivalent metalloids like arsenic, bt not other causes of protein misfolding. The key mediators of this response pathway are Cuz1, and the related but largely uncharacterized protein, Tmc1. We showed that Cuz1 functions as a novel zinc-dependent ubiquitin binding protein that interacts with the proteasome and the multifunctional chaperone Cdc48/p97. These data suggest a model in which Cuz1 protects cells from trivalent metalloids by recognizing misfolded proteins and delivering them to the proteasome for destruction. The long-term goal of my work is to provide a better understanding of how intracellular protein degradation occurs. In this application, I use a combination of biochemical, genetic, cell biologic, proteomic, and structural approaches to understand the stress response pathway mediated by Cuz1 and Tmc1. First, I will characterize the ubiquitin binding function of Cuz1. My data indicate a potentially novel evolutionarily conserved ubiquitin recognition motif within Cuz1. If verified, Cuz1 might represent the founding member of a new class of ubiquitin binding proteins. Second, I will utilize proteomics approaches to identify specific substrates of Cuz1. Because Cuz1 is remarkably specific in protecting cells from metalloid-induced proteotoxicity, these studies may provide insight into one of the most important questions in protein degradation, which relates to how specificity is generated and maintained. Third, we will undertake the first significant characterization of Tmc1, a zinc finger protein with sequence and phenotypic similarity to Cuz1. I will determine how Cuz1 and Tmc1 function in coordinating their protective stress response. Together, these aims will provide insight into a fundamental aspect of cell biology, regulated protein degradation, and in so doing will inform the many diseases associated with misfolded proteins.