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内容(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伙伴形成更大的寡聚组件 蛋白质，在这些复合体之外是不稳定的“孤儿”蛋白质。突变和基因突变的积累 肿瘤细胞进化过程中的染色体异常会产生一个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。 尽管普遍存在表现出协作稳定性的蛋白质，但质量控制机制 管理协作稳定性和促进孤儿蛋白的降解几乎完全是 哺乳动物身上没有特征的。我们的目标是确定调控的机制和细胞因子 协同蛋白质稳定性，并利用这些途径选择性地靶向具有不稳定蛋白质组的肿瘤。 我们已经确定HUWE1是一种泛素连接酶，它以未组装的孤儿蛋白为目标进行降解。我们的 假设HUWE1是一种关键的细胞因子，它介导孤儿蛋白的破坏并增强 在蛋白质组不稳定的非整倍体细胞中的适应性。为了探索这一假设，我们将：(1)执行结构 HUWE1的功能分析以确定HUWE1选择和靶向底物的机制；(2) 在正常细胞和异倍体细胞中鉴定HUWE1底物；(3)确定HUWE1的缺失或获得的影响 整倍体和非整倍体细胞对蛋白质动态平衡的作用和对蛋白质毒性应激的反应。我们 已经确定了以HUWE1依赖和独立的方式被破坏的孤儿蛋白。我们 将使用基于识别的衬底的光学报告系统，该系统依赖于协作稳定性来执行 基于CRISPR/Cas9的基因筛查以识别参与HUWE1依赖的未知因素 以及未组装的孤儿蛋白的独立降解。本提案中的研究成果将 确定以孤儿降解通路为靶点是否是降低肿瘤适合度的有效策略 具有不稳定蛋白质组的细胞。如果成功，我们将建立HUWE1利用的机制，以具体 靶向掺入的孤儿蛋白进行降解并确定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很敏感。我们还将从基因上定义孤儿 哺乳动物中的蛋白质降解途径。长期目标是开发分子策略，目标是 利用新的特性降低蛋白质稳态能力有限的细胞的适合度 在这项研究中确定了抑制有缺陷的孤儿蛋白破坏的控制机制。调查结果 拟议的研究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，可用于对抗与衰老相关的人类 病理学。

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.