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

项目总结 HSPA1a是一种应激诱导的70kodalton热休克蛋白，在肿瘤细胞中发挥重要作用 生死存亡。这种蛋白也定位于细胞表面和应激和癌症的细胞外介质。 细胞。HSPA1A膜阳性肿瘤对放射治疗具有抵抗力，表现出更高的侵袭性，以及 发生远处转移，而膜结合和细胞外HSPA1a发挥免疫刺激作用 和免疫抑制功能。最近的研究表明，HSPA1A的膜定位依赖于 关于其与特定脂类的选择性相互作用和蛋白质的胞外转运是由几种 机制包括外切体、分泌性溶酶体和脂筏。然而，HSPA1A的贩运 在细胞表面，膜结合型HSPA1a与胞外HSPA1a的关系以及 触发HSPA1A重新定位的特定脂类或蛋白质修饰仍不清楚。要回答这些问题 基本问题，我们建议表征HSPA1a膜的转运机制 通过确定脂质相互作用将HSPA1a募集到内体途径的机制来实现易位。 这一目标将通过确定HSPA1A在不同亚细胞中的重新定位模式来实现 热休克后应用亚细胞分离和成像的隔室，并耗尽特定的内吞液层 脂类与药物或脂类生物传感器。此外，我们将确定HSPA1A的细胞外转运 取决于它的膜定位和与特定脂类的结合。为此，我们将划定 膜结合型HSPA1a与胞外HSPA1a的关系及细胞外HSPA1A型检测 内切/外切途径抑制物和脂质生物传感器的存在或不存在。最后，我们将 表征HSPA1a上影响其转录后修饰的脂质成分变化 膜定位。为了确定这些变化，我们将表征脂质成分的动态 在应激细胞中，使用靶向脂质组学方法，并识别差异表达的脂质修饰 基因。此外，我们将确定HSPA1A上的特定翻译后修改是否 使用靶向蛋白质组学方法负责增加膜结合的HSPA1A。这项建议 将提供基本知识，形成未来控制膜的干预措施的基础 相关的和细胞外的HSPA1A旨在降低肿瘤的存活率和提高免疫反应。 此外，该项目将培养多个非传统和第一代本科生和硕士水平 学生，并将为他们在科学领域的不同职业做好准备。

PROJECT SUMMARY HSPA1A is a stress-inducible seventy-kilodalton heat shock protein that plays essential roles in tumor cell survival. This protein also localizes at the cell-surface and the extracellular medium of stressed and cancer cells. HSPA1A-membrane positive tumors are resistant to radiation therapy, show increased invasiveness, and develop distant metastasis, while membrane-bound and extracellular HSPA1A exert both immunostimulatory and immunosuppressive functions. Recent research revealed that HSPA1A's membrane localization depends on its selective interaction with specific lipids and the protein's extracellular transport is mediated by several mechanisms including exosomes, secretory lysosomes, and lipid rafts. However, the trafficking of HSPA1A towards the cell surface, the relationship between membrane-bound and extracellular HSPA1A, and the specific lipid or protein modifications that trigger HSPA1A's re-localization remain unknown. To answer these fundamental questions, we propose to characterize the trafficking mechanism of HSPA1A's membrane translocation by determining the mechanism by which lipid interactions recruit HSPA1A to endosomal pathway. This objective will be achieved by determining the re-localization pattern of HSPA1A in different subcellular compartments after heat shock using subcellular fractionation and imaging, and depleting specific endosomal lipids with drugs or lipid-biosensors. Furthermore, we will determine whether HSPA1A's extracellular transport depends on its membrane localization and binding to specific lipids. To this end, we will delineate the relationship between membrane-bound and extracellular HSPA1A, and then measure extracellular HSPA1A in the presence or absence of the endo/exosomal pathway inhibitors and lipid-biosensors. Lastly, we will characterize the lipid composition alterations and post-translational modifications on HSPA1A that affect its membrane localization. To determine these changes, we will characterize the dynamics of the lipid composition in stressed-cells, using a targeted lipidomics approach, and identify the differentially expressed lipid modifying genes. Additionally, we will determine whether specific post-translational modifications on HSPA1A are responsible for the increased membrane-bound HSPA1A using a targeted proteomics approach. This proposal will provide fundamental knowledge that will form the basis for future interventions to control membrane- associated and extracellular HSPA1A aiming to decrease tumor survival and increase the immune response. Furthermore, this project will train multiple non-traditional and first-generation undergraduate and master level students, and will prepare them for diverse careers in science.