Leveraging orphan protein degradation pathways to target cells with unstable proteomes

许多肿瘤细胞含有异常的染色体含量（非整倍体），这导致了 成千上万的蛋白质。这些蛋白质的一个子集通常与配偶体形成较大的寡聚体组装体 蛋白质和不稳定的“孤儿”蛋白质以外的这些复合物。突变的积累， 肿瘤细胞进化过程中的染色体异常产生越来越不稳定的蛋白质组， 提高了对蛋白质质量控制系统的需求，以选择性地去除这些不稳定的缺陷蛋白质。 尽管蛋白质普遍表现出协同稳定性，但质量控制机制， 控制协同稳定性和促进孤儿蛋白的降解几乎完全是 在哺乳动物中没有特征的。我们的目标是确定调节的机制和细胞因子， 协同蛋白质稳定性，并利用这些途径选择性靶向具有不稳定蛋白质组的肿瘤。 我们已经确定Huwe 1作为一种泛素连接酶，其靶向未组装的孤儿蛋白进行降解。我们 假设Huwe 1是介导孤儿蛋白破坏和增强免疫应答的关键细胞因子， 在具有不稳定蛋白质组的非整倍体细胞中的适应性。为了验证这一假设，我们将：（1）执行结构 对Huwe 1进行功能分析，以确定Huwe 1选择和靶向底物的机制;（2） 鉴定正常和非整倍体细胞中的Huwe 1底物;（3）确定Huwe 1缺失或获得的影响 在整倍体和非整倍体细胞中蛋白质稳态和对蛋白毒性应激的反应中起作用。我们 已经鉴定出孤儿蛋白，这些孤儿蛋白以Huwe 1依赖和独立的方式被破坏。我们 将利用基于依赖于协作稳定性的识别底物的光学报告系统来执行 基于CRISPR/Cas9的遗传筛选，以确定参与Huwe 1依赖性 和未组装的孤儿蛋白的独立降解。本提案中的研究成果将 确定靶向孤儿降解途径是否是降低肿瘤适应性的有效策略 蛋白质组不稳定的细胞如果成功，我们将建立Huwe 1利用的机制， 靶向掺入的孤儿蛋白以降解，并确定是否存在具有不稳定蛋白质组的非整倍体细胞 对孤儿蛋白降解途径中的缺陷敏感。我们还将从基因上定义孤儿 哺乳动物的蛋白质降解途径。长期目标是发展分子策略， 通过利用新表征的质量降低具有有限蛋白质稳态能力的细胞的适应性 在本研究中确定了抑制缺陷孤儿蛋白破坏的控制机制。这些发现 从拟议的研究中产生的结果将为我们识别细胞的目标提供实质性的进展， 调节蛋白质质量控制的机制，可用于对抗与人类衰老相关的疾病。 病理

Many tumor cells contain abnormal chromosome content (aneuploidy), which results in altered expression of thousands of proteins. A subset of these proteins normally form larger oligomeric assemblies with partner proteins and are unstable as “orphan” proteins outside of these complexes. The accumulation of mutations and chromosomal abnormalities during tumor cell evolution generates an increasingly unstable proteome that elevates the need for protein quality control systems to selectively remove these unstable defective proteins. Despite the prevalence of proteins that demonstrate collaborative stability, the quality control mechanisms that govern collaborative stability and facilitate the degradation of orphan proteins is almost entirely uncharacterized in mammals. Our objective is to determine the mechanisms and cellular factors that regulate collaborative protein stability and utilize these pathways to selectively target tumors with unstable proteomes. We have identified Huwe1 as a ubiquitin ligase that targets unassembled orphan proteins for degradation. Our hypothesis is that Huwe1 is a critical cellular factor the mediates orphan protein destruction and enhances fitness in aneuploid cells with unstable proteomes. To probe this hypothesis, we will: (1) perform structure function analysis on Huwe1 to determine the mechanism of how Huwe1 selects and targets substrates; (2) identify Huwe1 substrates in normal and aneuploid cells; (3) determine the impact of loss or gain of Huwe1 function on protein homeostasis and the response to proteotoxic stress in euploid and aneuploid cells. We have identified orphan proteins that are destroyed in both a Huwe1-dependent and independent manner. We will utilize optical reporter systems based on identified substrates that rely on collaborative stability to perform CRISPR/Cas9-based genetic screens to identify unknown factors that participate in both Huwe1-dependent and independent degradation of unassembled orphan proteins. Research outcomes within this proposal will establish if targeting orphan degradation pathways represents an effective strategy to reduce fitness in tumor cells with unstable proteomes. If successful, we will establish the mechanism Huwe1 utilizes to specifically target incorporated orphan proteins for degradation and determine if aneuploid cells with unstable proteomes are sensitive to defects in orphan protein degradation pathways. We will also genetically define the orphan protein degradation pathway in mammals. The long-term goal is to develop molecular strategies aimed at reducing fitness in cells with limited protein homeostasis capacity by utilizing newly characterized quality control mechanisms identified during this research to inhibit defective orphan protein destruction. The findings resulting from the proposed research will provide substantial progress toward our goal of identifying cellular mechanisms that regulate protein quality control that can be leveraged to combat aging associated human pathology.