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Naturally occurring mutations alter the function of HSPA1A , a key regulator of the cellular stress response in humans
Author(s) -
Oliverio Ryan Michael,
Hess Kyle,
Nikolaidis Nikolas
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.811.4
Subject(s) - biology , microbiology and biotechnology , mutant , mutagenesis , heat shock , heat shock protein , clpb , mutation , gene , function (biology) , genetics , stress granule , green fluorescent protein , translation (biology) , messenger rna
An essential question in molecular evolution is how mutations enable organisms to adapt and survive their environments. At the cellular level, this same question relates to the ability of cells to adapt and survive stressful conditions, such as disease. Cellular adaptation to stress is regulated and maintained by the cellular stress response system. Molecular chaperones, and, in particular the 70‐kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system, and alterations in their function have direct physiological consequences. Therefore, it is of vital importance to determine how natural mutations alter the function of these proteins and how such changes affect cellular and organismal adaptation. To answer this question we tested whether natural single nucleotide polymorphisms (SNPs) found on HSPA1A , the major stress inducible Hsp70 gene in humans, alter protein 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 first determined whether any of these mutations affected the intracellular localization of HSPA1A within mammalian cells. These experiments were performed by tagging HSPA1A to GFP, using fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, to view where the proteins localize via confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Next, 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 two of the mutations caused increased cell death as compared to the WT, suggesting that some mutations alter the chaperone function of HSPA1A. Furthermore, live‐dead assays showed that human cells carrying some of these mutations had significantly less resistance to heat stress than cells expressing the WT protein. Given that these natural variants are either population‐specific or clinical we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress and adapt to environmental perturbations.