NIH DIRECTOR'S PIONEER AWARD

美国国立卫生研究院院长先锋奖

• 批准号: 7201731
• 负责人: LILA M GIERASCH
• 金额: $ 78.5万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-28 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7201731
• 关键词: NIH Roadmap Initiative tag; bacteria; bioimaging /biomedical imaging; cell free system; chemical stability; evolution; fluorescence microscopy; intracellular; method development; molecular /cellular imaging; nuclear magnetic resonance spectroscopy; protein biosynthesis; protein folding; protein structure function

Surprisingly little is known about how proteins fold in vivo, yet it is this process, and not the test-tube idealized folding reaction so intensively studied over the past several decades, that is crucial to the fitness of an organism. The fidelity of folding and the stability of proteins in the cell are critical to their functions, their degradation, and their vulnerability to aggregation. Many diseases are now known to arise from defects in protein folding, either because of the loss or alteration of essential protein functions, or because of the build-up of toxic species such as aggregates. We believe that novel methods and creative collaborations will allow us to overcome the daunting technical obstacles that have impeded progress on protein folding in the cell. Focusing first on a small model protein for which we have detailed descriptions of folding in vitro will enable methods optimization. Folding in cellular conditions will be followed in systems of increasing complexity: bacterial protein expression, cell-free biosynthesis, and semi-permeabilized or intact eukaryotic cell expression. The new strategies will reveal how larger proteins of biomedical interest adopt their structures in their cellular context and how this process may go awry. Methodologically, we anticipate placing major reliance on spectroscopic methods, including fluorescence and nuclear magnetic resonance, and using novel labeling strategies to observe the protein under study in the complex cellular milieu. Complementary in-cell imaging methods will be used to insure that observed signals report on relevant phenomena and to reveal novel functionally significant spatial localization patterns. We anticipate that this research will lead to new paradigms for how amino acid sequences encode folding information and that the resulting enhanced understanding of folding in vivo will lead to new strategies for therapeutic intervention in misfolding and aggregation diseases.

令人惊讶的是，人们对蛋白质在体内是如何折叠的知之甚少，但这是一个过程，而不是试管 在过去的几十年里，理想化的折叠反应得到了广泛的研究，这对 一个有机体的健康。细胞中蛋白质的折叠保真度和稳定性对它们的功能至关重要。 功能，其退化，以及它们对聚集的脆弱性。现在已知许多疾病 由蛋白质折叠缺陷引起，无论是由于必需蛋白质的丢失还是改变 功能，或因为积累的有毒物质，如聚集。我们相信这本小说 方法和创造性的合作将使我们能够克服令人生畏的技术障碍， 阻碍了细胞内蛋白质折叠的进展。首先关注一个小的模型蛋白， 我们对体外折叠的详细描述将使方法优化成为可能。细胞内折叠 条件将遵循日益复杂的系统：细菌蛋白质表达，无细胞 生物合成和半透化或完整真核细胞表达。新战略将 揭示了生物医学感兴趣的较大蛋白质如何在细胞环境中采用它们的结构，以及它们是如何在细胞中形成的。 这个过程可能会出错。从方法上讲，我们预计将主要依赖光谱 方法，包括荧光和核磁共振，并使用新的标记策略， 在复杂的细胞环境中观察所研究的蛋白质。互补细胞内成像 方法将被用来确保观察到的信号报告相关的现象，并揭示新的 功能显著的空间定位模式。我们预计，这项研究将导致新的 氨基酸序列如何编码折叠信息的范例，以及由此产生的增强 对体内折叠的理解将导致对错误折叠的治疗干预的新策略， 聚集性疾病