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

项目摘要 热休克蛋白70与细胞膜之间的相互作用是一种新的和很大程度上未表征的功能， 这些不可或缺的分子伴侣。我们假设热休克蛋白70和脂质之间的相互作用是 这是它们膜相关功能的关键步骤，脂质结合为它们提供了必要的 在细胞应激和疾病状态下， 癌热休克蛋白70与脂质的相互作用抑制肿瘤生长，诱导细胞死亡，激活肿瘤细胞， 免疫系统，稳定膜，调节营养循环（微自噬）。这种相互作用 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在其脂质结合功能方面是不同的。但是，在这方面， Hsp70与人类细胞中脂质相互作用的条件以及负责的氨基酸残基 大部分仍是未知的。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（应激诱导的Hsp70）与HspA1A相互作用的条件。 人类，有脂质。对于这项任务，我们将使用几种人类细胞系，它们将受到不同的处理。 改变膜脂质组成的治疗。HspA1A与脂质的相互作用将使用 下拉测定，在存在或不存在已知脂质结合蛋白和荧光标记的情况下的细胞成像， 脂质、亚细胞分级分离和细胞表面生物素化。此外，靶向脂质组学方法将 用于鉴定HspA1A天然脂质配体。其次，我们将鉴定和表征氨基酸 介导HspA1A与脂质结合的分子机制。对于这项任务， 几种氨基酸，这将是预测使用计算技术和观察从 文学，将被颠覆。突变对重组HspA1A与脂质结合的影响将是 使用脂质体沉降法和表面等离子体共振光谱定量。 此外，通过评估突变对HspA1A功能和稳定性的影响， 伴侣的功能。最后，突变对HspA1A的脂质结合特性的影响将是 使用荧光标记的HspA1A和下拉测定，成像， 和亚细胞分级分离。这项提案将提供基本知识，使我们能够测试 在人类细胞中的功能丧失突变的影响，并确定其生理意义。如果验证人 通过这些实验，我们的脂质结合特异性假说将使我们能够进一步阐明这种新的性质 热休克蛋白70 s，这与细胞应激反应，膜生物学和疾病的关键关联 比如癌症此外，该项目将培养多个非传统和第一代 本科和硕士水平的学生，并将准备他们进入公司，研究实验室， 先进的学术课程。

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.