Localization of Heat Shock Proteins HSPA6 (Hsp70B′), HSPA1A (Hsp70-1) and HSPA8 (Hsc70) in Cultured Human Neuronal Cells

Heat shock proteins (Hsps) are protein repair agents that detect and refold misfolded proteins and prevent their aggregation. Accumulation of protein aggregates is a key feature of neurodegenerative diseases. Treatments that showed beneficial effects in current animal models of neurodegenerative diseases have repeatedly failed in human clinical trials. A better understanding of the proteostasis machinery in human cells is required to design more effective therapeutic compounds. HSPA6 (Hsp70B′) is a stress-inducible member of the HSPA (Hsp70) multigene family that is absent in rat and mouse genomes and has been little studied compared to inducible HSPA1A (Hsp70-1), or constitutively expressed HSPA8 (Hsc70). HSPAs are involved in refolding proteins that misfold due to stress or disease. Binding to DNAJ (Hsp40) inhibits aggregation of misfolded proteins until they can be refolded by HSPA, but this machine cannot dissociate aggregated proteins. Recent studies have identified a disaggregase function performed by the mammalian HSPH (Hsp110) family in co-operation with HSPA/DNAJ in vitro. To advance knowledge of the mammalian disaggregation/refolding machine, this thesis investigates localization of HSPA6, HSPA1A, and HSPA8 with other components of the machine following stress in differentiated human neuronal SH-SY5Y cells. Unique targeting of HSPA6 to nuclear perispeckles and differential localization with disaggregation/refolding machine components at the nucleolus compared to HSPA1A or HSPA8 is observed following thermal stress. siRNA knockdown demonstrates that HSPA6 is protective in the neuronal stress response. MG132 proteotoxicity induces HSPA6 which targets the periphery of cytoplasmic protein aggregates with components of the disaggregation/refolding machine and thus may be involved in the response of differentiated neuronal cells to protein aggregation. Constitutively expressed HSPA8 exhibits similar intracellular targeting as inducible HSPA1A and may act as a rapid stress responder in neuronal cells, circumventing the time lag required to upregulate HSPA1A. In cancer cells, elevated levels of Hsps contribute to their ability to resist cell death. The chemotherapeutic agent cisplatin induces components of the disaggregation/refolding machine. Knockdown of HSPA family members enhances the killing effect of cisplatin on human neuroblastoma cells.Ph.D