Leveraging orphan protein degradation pathways to target cells with unstable proteomes

利用孤儿蛋白降解途径靶向具有不稳定蛋白质组的细胞

• 批准号: 10004157
• 负责人: Eric J Bennett
• 金额: $ 30.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-17 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004157
• 关键词: Aging; Aneuploid Cells; Aneuploidy; Base Pairing; Binding Proteins; Bioinformatics; CRISPR/Cas technology; Cell Line; Cells; Chromosomal Duplication; Chromosomal Insertion; Chromosome Deletion; Chromosome abnormality; Complex; DNA Sequence Alteration; Data; Defect; Degradation Pathway; Event; Evolution; Flow Cytometry; Future; Genetic; Genetic Screening; Genome; Genomic Instability; Genomics; Goals; Homeostasis; Human; Human Pathology; Impairment; Malignant Neoplasms; Mammals; Mediating; Methods; Molecular; Mutation; Normal Cell; Oncogenes; Optical reporter; Orphan; Outcomes Research; Pathway interactions; Prevalence; Proteins; Proteome; Proteomics; Quality Control; Research; Stream; Stress; Structure; System; Testing; Therapeutic; Transcriptional Activation; Tumor Suppressor Proteins; Ubiquitin; base; cancer cell; cell growth; combat; fitness; genome-wide; in vivo; inhibitor/antagonist; insertion/deletion mutation; interest; loss of function; man; multicatalytic endopeptidase complex; neoplastic cell; novel therapeutics; overexpression; protein degradation; proteostasis; proteotoxicity; public health relevance; response; small hairpin RNA; stressor; tumor; tumorigenesis; ubiquitin ligase; wasting

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.

许多肿瘤细胞含有异常的染色体含量（非整倍性），从而改变了 数千种蛋白质。这些蛋白质的一个子集通常与伴侣形成较大的寡聚组件 蛋白质在这些复合物之外的“孤儿”蛋白质不稳定。突变的积累和 肿瘤细胞演化期间染色体异常会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，以选择性去除这些不稳定的缺陷蛋白质。 尽管蛋白质的流行率证明了协作稳定性，但质量控制机制， 管理协作稳定性并促进孤儿蛋白的降解几乎完全是 在哺乳动物中未表征。我们的目标是确定调节的机制和细胞因子 协作蛋白质稳定性，并利用这些途径来选择性地靶向具有不稳定蛋白质组的肿瘤。 我们已经将HUWE1识别为一种泛素连接酶，它靶向未组装的孤儿蛋白以降解。我们的 假设是Huwe1是介导孤儿蛋白破坏并增强的关键细胞因子 具有不稳定蛋白质组的非整倍体细胞中的适应性。为了探究这一假设，我们将：（1）执行结构 HUWE1上的功能分析，以确定HUWE1如何选择和目标底物的机制； （2） 鉴定正常和非整倍体细胞中的HuWe1底物； （3）确定HUWE1损失或增益的影响 蛋白质稳态的功能以及对蛋白质毒性应激的反应，在蛋白质毒性胁迫和非整倍体细胞中的作用。我们 已经确定了以Huwe1依赖性和独立方式破坏的孤儿蛋白。我们 将基于依赖协作稳定性的确定基材使用光学记者系统 基于CRISPR/CAS9的遗传筛选以识别参与HUWE1依赖性的未知因素 并独立降解未组装的孤儿蛋白。该提案中的研究成果将 确定靶向孤儿降解途径是否代表了降低肿瘤适应性的有效策略 具有不稳定蛋白质组的细胞。如果成功，我们将建立Huwe1使用的机制 靶标并掺入孤立蛋白进行降解，并确定是否具有不稳定蛋白质组的非整倍型细胞 我们还将基因定义孤儿 哺乳动物中的蛋白质降解途径。长期目标是制定针对的分子策略 通过利用新特征的质量来降低具有有限蛋白质稳态能力的细胞的适应性 在本研究中确定的控制机制抑制孤儿蛋白破坏的缺陷。发现 拟议的研究产生的将为我们识别细胞的目标提供重大进展 调节蛋白质质量控​​制的机制，可以利用与衰老相关的人 病理。