Functional characterization of natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible 70kDa heat shock gene in humans

The cellular stress response system is responsible for maintaining cellular homeostasis during periods of stress caused by environmental insults including pathogens and disease. Molecular chaperones, and, in particular the 70‐kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system. Therefore, we hypothesized that natural occurring variations in the sequence of Hsp70s will affect cellular and subsequently species adaptation. To investigate the relationship between adaptation, disease susceptibility, and Hsp70s' variation we tested whether natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible Hsp70 gene in humans, alter its function. Specifically, the wild‐type human HSPA1A sequence was subcloned into mammalian expression vectors, and the mutated gene variants were generated using site‐directed mutagenesis. We then determined whether any of these mutations affected the intracellular localization of HSPA1A. These experiments were performed by using GFP‐tagged HSPA1A and fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, and visualized using confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Furthermore, we determined whether the mutations affected the ability of HSPA1A to prevent cell death by inhibiting the formation of protein aggregates caused by poly‐glutamine carrying huntingtin proteins. This assay determined that one of the mutations caused increased cell death as compared to the WT, suggesting that it may alter HSPA1A's ability to prevent protein aggregation. We also determined whether these mutations affect the ability of HSPA1A to refold heat‐denatured luciferase. These assays revealed that only one mutation resulted in significantly lower levels of refolded enzyme, suggesting a putative loss‐of‐chaperone function. Lastly, live‐dead assays revealed that human cell lines carrying two of these mutations had significantly less resistance to heat and ethanol stress than cells expressing the WT protein. Given that these natural variants are found at a very low frequency in humans we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress. Support or Funding Information This project was supported by funds from NIH and CSUF to NN