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

项目总结/摘要 尽管经过几十年的研究，蛋白质如何在细胞中折叠仍然知之甚少。蛋白质折叠和错误折叠 是从癌症到神经变性等疾病的发病机制的基础。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这种复合物。 细胞内环境，包括分子伴侣、膜和其他生物分子。此外，我们目前 体外和体内折叠的知识主要依赖于低通量的单蛋白实验。 虽然提供了大量的细节，但这些方法不能同时测试差异折叠和错误折叠是如何发生的。 影响疾病生理学。超过20%的人类蛋白质含有二硫键， 这些键的形成典型地代表了在氧化条件下获得天然折叠的限速步骤 条件因此，监测天然二硫键形成的动力学可以提供以下方面的替代： 成功的蛋白质折叠（Mamathambika和Bardwell，2008）。我们的团队率先使用有针对性的 质谱法来监测细胞蛋白质合成。在这里，我们提出了一种全新的方法， 同时监测数百种蛋白质的氧化蛋白质折叠：使用靶向定量质量 使用质谱法监测体内天然二硫键形成的动力学。在我的小组里， 专注于多发性骨髓瘤的研究，这是一种浆细胞恶性血液病，目前尚无治愈方法。这 疾病从根本上说是一种异常蛋白质稳态的紊乱：据认为， 免疫球蛋白的增加导致许多已知的临床后遗症，同时通过增加免疫球蛋白的含量来诱导细胞凋亡。 未折叠蛋白质应激是一线治疗策略。在这里，我们将首先开发生物化学和 蛋白质组学工具，以监测天然二硫键形成的nascently合成的蛋白质内 内质网然后，我们将使用这些工具，结合调整免疫球蛋白 通过CRISPR抑制和激活来合成，以测试骨髓瘤细胞 由于未折叠蛋白质应激增加，对蛋白酶体抑制非常敏感。最后，我们将测试 氧化折叠分子伴侣的调节对许多类别的同时折叠动力学的影响 骨髓瘤相关的分泌和细胞外蛋白。我们预计这些实验方法 将为我们对体内整体蛋白质折叠的理解提供重大进展，从而解决 这是我们对这一重要生物学过程的认识中的一个重大空白。此外，我们的结果将提供一个 突破了对广泛的细胞内蛋白质动力学的研究，这是无法用 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.