Interaction between HspA1A, a seventy-kDa heat shock protein, and lipids in stressed cells

项目概要 Hsp70 与细胞膜之间的相互作用是一种新的、很大程度上未被表征的功能。 这些不可或缺的分子伴侣。我们假设 Hsp70 与脂质之间的相互作用是 这是其膜相关功能的关键一步，并且脂质结合为它们提供了必要的 在细胞应激和疾病条件下，在不同膜上定位和发挥作用的特异性 癌症。 Hsp70 与脂质的相互作用抑制肿瘤生长、诱导细胞死亡、激活 免疫系统、稳定膜并调节营养循环（微自噬）。这次互动 取决于脂质环境，由多种类型的分子力介导，并被改变 核苷酸结合。此外，Hsp70 的脂质结合功能也有所不同。然而， Hsp70 与人体细胞中的脂质相互作用的条件以及负责的氨基酸残基 对于结合仍然大多未知。为了回答这两个基本问题，我们提出了两个具体的建议 目标。首先，我们将确定有利于 HspA1A（应激诱导型 Hsp70）相互作用的条件。 人类，与脂质。对于这项任务，我们将使用几种人类细胞系，这些细胞系将受到不同的处理 改变膜脂成分的治疗。 HspA1A 与脂质的相互作用将使用以下方法进行评估 Pull-down 测定、存在或不存在已知脂质结合蛋白和荧光的细胞成像 脂质、亚细胞分级分离和细胞表面生物素化。此外，有针对性的脂质组学方法将 可用于验证 HspA1A 天然脂质配体。其次，我们将鉴定和表征氨基酸 介导 HspA1A-脂质结合，并阐明这种相互作用的分子机制。对于这个任务， 几种氨基酸，将使用计算技术和观察结果进行预测 文学，将会变异。重组 HspA1A 与脂质结合的突变效应将是 使用脂质体沉降法和表面等离子体共振光谱法进行定量。 此外，突变对 HspA1A 功能和稳定性的影响将通过评估改变来确定 的陪伴功能。最后，突变对 HspA1A 脂质结合特性的影响将是 使用荧光标记的 HspA1A 并结合 Pull-down 测定、成像、 和亚细胞分级分离。该提案将提供基础知识，使我们能够测试 人类细胞功能丧失突变的影响并确定其生理影响。如果通过验证 这些实验，我们的脂质结合特异性假设将使我们能够进一步阐明这种新特性 Hsp70 的组成，与细胞应激反应、膜生物学和疾病密切相关 癌症等疾病。此外，该项目还将培训多名非传统和第一代 本科生和硕士水平的学生，将为他们进入公司、研究实验室和 高级学术课程。

PROJECT SUMMARY The interaction between Hsp70s and cellular membranes is a new and largely uncharacterized function of these indispensable molecular chaperones. We hypothesize that the interaction between Hsp70s and lipids is a critical step for their membrane-associated functions, and that lipid-binding provides them with the necessary specificity to localize and function at different membranes during cellular stress and disease conditions like cancer. The interaction of Hsp70s with lipids suppresses tumor growth, induces cell death, activates the immune system, stabilizes membranes, and regulates nutrient recycling (microautophagy). This interaction depends on the lipid environment, is mediated by multiple types of molecular forces, and is altered by nucleotide binding. Furthermore, Hsp70s are differentiated with respect to their lipid-binding function. However, the conditions under which Hsp70s interact with lipids in human cells and the amino acid residues responsible for binding remain mostly unknown. To answer these two fundamental questions we propose two specific aims. First, we will determine the conditions that favor the interaction of HspA1A, the stress-inducible Hsp70 in humans, with lipids. For this task we will use several human cell lines, which will be subjected to different treatments that alter membrane lipid composition. The interaction of HspA1A with lipids will be assessed using pull-down assays, cellular imaging in the presence or absence of known lipid-binding proteins and fluorescent lipids, subcellular fractionation, and cell surface biotinylation. Furthermore, a targeted lipidomics approach will be used to authenticate HspA1A native lipid ligands. Second, we will identify and characterize the amino acids that mediate the HspA1A-lipid binding, and elucidate the molecular mechanism of this interaction. For this task, several amino acids, which will be predicted using computational techniques and observations from the literature, will be mutated. The mutational effect on the binding of recombinant HspA1A to lipids will be quantified using the liposome sedimentation method and Surface Plasmon Resonance spectroscopy. Additionally, the mutational effect on HspA1A function and stability will be determined by assessing alterations of the chaperone function. Finally, the mutational effect on the lipid-binding properties of HspA1A will be verified in human cells using fluorescently labeled HspA1A and a combination of pull-down assays, imaging, and subcellular fractionation. This proposal will provide fundamental knowledge that will allow us to test the effects of loss-of-function mutations in human cells and identify their physiological implications. If validated by these experiments, our lipid-binding specificity hypothesis will allow us to further elucidate this novel property of Hsp70s, which has critical associations with the cellular stress response, membrane biology, and disease conditions like cancer. Furthermore, this project will train multiple non-traditional and first-generation undergraduate and master level students, and will prepare them to enter companies, research labs, and advanced academic programs.