In vivo monitoring of oxidative protein folding through time-resolved quantitative mass spectrometry

通过时间分辨定量质谱法体内监测氧化蛋白折叠

• 批准号: 9167306
• 负责人: Arun P. Wiita
• 金额: $ 189万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9167306
• 关键词: Address; Apoptosis; Biochemical; Biological Process; Biology; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Disease; Endoplasmic Reticulum; Environment; Extracellular Protein; Heat shock proteins; Hematologic Neoplasms; Hematopoietic Neoplasms; Homeostasis; Human; Immunoglobulins; In Vitro; Kinetics; Knowledge; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Methods; Modeling; Molecular Chaperones; Monitor; Multiple Myeloma; Nerve Degeneration; Pathogenesis; Physiology; Plasma Cells; Production; Proteasome Inhibition; Protein Biosynthesis; Protein Dynamics; Proteins; Proteome; Proteomics; Proxy; Testing; Therapeutic; Time; clinically relevant; disulfide bond; in vivo; killings; neoplastic cell; novel strategies; protein folding; protein misfolding; research study; stress protein; tool

PROJECT SUMMARY/ABSTRACT Despite decades of study, how proteins fold in cells remains poorly understood. Protein folding and misfolding underlies the pathogenesis of diseases ranging from cancer to neurodegeneration. Much of what we do know about protein folding has been gathered from in vitro experiments, which do not fully model the complex intracellular environment including chaperones, membranes, and other biomolecules. Furthermore, our current knowledge of folding, both in vitro and in vivo, primarily relies on low-throughput, single-protein experiments. While providing great detail, these methods cannot simultaneously test how differential folding and misfolding across the proteome impacts disease physiology. Over 20% of human proteins contain disulfide bonds, and formation of these bonds typically represents the rate-limiting step in achieving the native fold under oxidizing conditions. Therefore, monitoring the kinetics of native disulfide bond formation can provide a proxy for successful protein folding (Mamathambika and Bardwell, 2008). Our group has pioneered the use of targeted mass spectrometry to monitor cellular protein synthesis. Here, we propose an entirely new approach to monitor oxidative protein folding across hundreds of proteins simultaneously: using targeted, quantitative mass spectrometry to monitor the kinetics of native disulfide bond formation in vivo. In my group we specifically focus on the study of multiple myeloma, a hematologic malignancy of plasma cells with no known cure. This disease is fundamentally a disorder of aberrant protein homeostasis: it is thought that unregulated production of immunoglobulin leads to many of the known clinical sequelae, while inducing apoptosis by increasing unfolded protein stress is a first-line therapeutic strategy. Here, we will first develop biochemical and proteomic tools to monitor native disulfide bond formation in nascently synthesized proteins within the endoplasmic reticulum. We will then use these tools, in combination with tuning of immunoglobulin protein synthesis through CRISPR inhibition and activation, to test the clinically-relevant hypothesis that myeloma cells are exquisitely sensitive to proteasome inhibition due to increased unfolded protein stress. Finally, we will test the effects of modulation of oxidative folding chaperones on simultaneous folding kinetics across many classes of myeloma-relevant secreted and extracellular proteins. We anticipate that these experimental approaches will provide a significant advance toward our understanding of global protein folding in vivo, thereby addressing a major gap in our knowledge of this central biological process. Furthermore, our results here will provide a breakthrough toward the study of a broad range of intracellular protein dynamics that are inaccessible with other methods.

项目摘要/摘要 尽管进行了数十年的研究，但细胞中的蛋白质如何折叠仍然很少了解。蛋白质折叠和错误折叠 构成了从癌症到神经变性的疾病的发病机理。我们所知道的很多 关于蛋白质折叠已从体外实验中收集，这些实验并未完全模拟复合物 细胞内环境，包括伴侣，膜和其他生物分子。此外，我们的当前 在体外和体内折叠的知识主要依赖于低通量的单蛋白实验。 在提供大量细节的同时，这些方法无法同时测试差折叠和错误折叠的方式 跨蛋白质组会影响疾病的生理。超过20％的人蛋白包含二硫键，并且 这些键的形成通常代表在氧化下实现天然折叠的速率限制步骤 状况。因此，监测天然二硫键形成的动力学可以为 成功的蛋白质折叠（Mamathambika和Bardwell，2008年）。我们的小组率先使用了目标 质谱法监测细胞蛋白合成。在这里，我们提出了一种全新的方法 同时监测氧化蛋白在数百种蛋白质上折叠：使用靶向定量质量 光谱法监测体内天然二硫键形成的动力学。在我的小组中，我们特别 重点研究多发性骨髓瘤，这是浆细胞的血液系统恶性肿瘤，但没有已知的治愈方法。这 从根本上讲，疾病是一种异常蛋白稳态的疾病：人们认为生产不受管制 免疫球蛋白导致许多已知的临床后遗症，同时通过增加而诱导凋亡 展开的蛋白质应激是一线治疗策略。在这里，我们将首先开发生化和 蛋白质组学工具以监测本地合成的蛋白质中的天然二硫键形成 内质网。然后，我们将使用这些工具，结合对免疫球蛋白蛋白的调整 通过CRISPR抑制和激活的合成，以检验临床上与骨髓瘤细胞相关的假设 由于增加的蛋白质胁迫增加，对蛋白酶体抑制非常敏感。最后，我们将测试 氧化折叠伴侣调节对许多类别的同时折叠动力学的影响 与骨髓瘤相关的分泌和细胞外蛋白。我们预计这些实验方法 将为我们对体内全球蛋白质折叠的理解提供重大进步，从而解决 我们对这个中心生物学过程的了解的主要差距。此外，我们在这里的结果将提供 在研究广泛的细胞内蛋白质动力学方面的突破，这些动力学无法访问 其他方法。