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

项目摘要 HSPA 1A是一种应激诱导的热休克蛋白，在肿瘤细胞中起重要作用 生存这种蛋白质也定位于细胞表面和细胞外介质的压力和癌症 细胞HSPA 1A膜阳性肿瘤对放射治疗有抵抗力，表现出侵袭性增加，并且 发生远处转移，而膜结合和细胞外HSPA 1A发挥免疫刺激作用， 和免疫抑制功能。最近的研究表明，HSPA 1A的膜定位依赖于 它与特定脂质的选择性相互作用和蛋白质的细胞外运输是由几个介导的 机制包括外泌体、分泌溶酶体和脂筏。然而，HSPA 1A的贩运 对细胞表面，膜结合和细胞外的HSPA 1A之间的关系，和 触发HSPA 1A重新定位的特定脂质或蛋白质修饰仍然未知。回答这些 的基本问题，我们建议表征HSPA 1A的膜的运输机制 通过确定脂质相互作用将HSPA 1A募集到内体途径的机制来确定HSPA 1A的易位。 这一目标将通过确定HSPA 1A在不同亚细胞中的重新定位模式来实现 使用亚细胞分级分离和成像，以及消耗特定的内体， 脂质与药物或脂质生物传感器。此外，我们将确定HSPA 1A的细胞外转运是否 取决于其膜定位和与特定脂质的结合。为此，我们将描绘 膜结合和细胞外HSPA 1A之间的关系，然后测量细胞外HSPA 1A， 存在或不存在内/外泌体途径抑制剂和脂质生物传感器。最后，我们将 表征HSPA 1A上的脂质组成改变和翻译后修饰， 膜定位为了确定这些变化，我们将描述脂质组成的动态特性 在应激细胞中，使用靶向脂质组学方法， 基因.此外，我们还将确定HSPA 1A上的特异性翻译后修饰是否是 负责增加膜结合HSPA 1A使用靶向蛋白质组学方法。这项建议 将提供基础知识，为未来的干预措施，以控制膜- 相关的和细胞外的HSPA 1A，旨在减少肿瘤存活和增加免疫应答。 此外，该项目将培养多名非传统和第一代本科和硕士水平的 学生，并将为他们在科学的各种职业做好准备。

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.