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

项目摘要/摘要 尽管经过了几十年的研究，但蛋白质在细胞中如何折叠仍然知之甚少。蛋白质折叠与错误折叠 是从癌症到神经变性等各种疾病的基础。我们所知道的大部分 关于蛋白质折叠的研究是从体外实验中收集的，这些实验并没有完全模拟复杂的蛋白质 细胞内环境，包括伴侣、膜和其他生物分子。此外，我们目前的 对折叠的了解，无论是在体外还是在体内，主要依赖于低通量的单蛋白实验。 在提供大量细节的同时，这些方法不能同时测试差异折叠和错误折叠 整个蛋白质组影响疾病生理学。超过20%的人类蛋白质含有二硫键，并且 这些键的形成通常代表在氧化条件下实现自然折叠的限速步骤 条件。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(Mamathbika和Bardwell，2008)。我们的团队已经率先使用了靶向 用于监测细胞蛋白质合成的质谱仪。在这里，我们提出了一种全新的方法来 同时监测数百种蛋白质的氧化蛋白质折叠：使用目标、定量的质量 光谱监测体内天然二硫键形成的动力学。在我的团队里，我们特别 专注于多发性骨髓瘤的研究，多发性骨髓瘤是一种血液系统的浆细胞恶性肿瘤，目前尚无治愈方法。这 疾病从根本上说是一种异常的蛋白质动态平衡失调：人们认为，不受调控的生产 免疫球蛋白的增加会导致许多已知的临床后遗症，同时通过增加 未折叠蛋白质应激是一线治疗策略。在这里，我们将首先发展生化和 蛋白质组学工具监测新生合成的蛋白质中天然二硫键的形成 内质网。然后我们将使用这些工具，结合免疫球蛋白的调节 通过抑制和激活CRISPR合成，以检验骨髓瘤细胞的临床相关假设 对蛋白酶体的抑制非常敏感，这是由于增加的未折叠蛋白压力造成的。最后，我们将测试 氧化型折叠伴侣蛋白的调控对多类分子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，这些实验性的方法 将为我们理解体内的全球蛋白质折叠提供重大进展，从而解决 这是我们对这一中心生物过程的认识上的一大空白。此外，我们在这里的结果将提供一个 对广泛的细胞内蛋白质动力学的研究取得突破，这些动力学是无法用 其他方法。

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.