DELINEATING THE SECRETORY PATHWAY OF HSPA1A AFTER HEAT-SHOCK

Extracellular HSPA1A (eHSPA1A), the stressed-induced member of the HSP70 molecular chaperone family, regulates many vital processes, as it activates the innate and adaptive immune responses and promotes tumor cell death and suppression. Additionally, membrane-bound HSPA1A marks several types of cancer cells, making them resistant to therapy and radiation. Although HSPA1A translocates and anchors at the plasma membrane (PM) and is actively secreted from viable cells, it lacks known transmembrane domains, lipid-binding domains, and signal peptides. However, the relationship between membrane localization and secretion of HSPA1A remains largely undefined. Clarifying this relationship is vital for understanding HSPA1A’s function in tumor progression and metastasis. I hypothesized that the lipid-driven translocation of HSPA1A to the PM is the intermediate step of its secretion to the extracellular medium. To test my hypothesis, I monitored the secretion of HSPA1A after heat-shock in the presence and absence of specific lipid biosensors that will manipulate different membrane phospholipids. My results show that eHSPA1A increases continuously during the cells’ recovery after mild non-apoptotic heat shock. Furthermore, eHSPA1A is found exclusively in extracellular vesicles (EVs) under these conditions. Additionally, PM localized HSPA1A increased during recovery from heat shock, reached a maximal at 8 hours post-stress, and then declined. Depleting a specific lipid target via co-transfection with lipid biosensors resulted in inhibition of HSPA1A’s PM localization but did not affect the EM export of the protein via EVs. Both the timing and lipid-depletion support the notion that HSPA1A’s PM localization and EM export are independent and probably sequential events. My research provides new information on the mechanism of HSPA1A’s secretion and its relationship to lipid-binding, which will be used in future research to define the mechanistic details and determine what recruits HSPA1A for EM export in a form that can activate the immune system against tumor cells.