75-kDa glucose-regulated protein (GRP75) is a novel molecular signature for heat stress response in avian species

Abstract

Glucose-regulated protein 75 (GRP75) was first characterized in mammals as a heat shock protein-70 (HSP70) family stress chaperone based on its sequence homology. Extensive studies in mammals showed that GRP75 is induced by various stressors such as glucose deprivation, oxidative stress, and hypoxia, although it remained unresponsive to the heat shock. Such investigations are scarce in avian (nonmammalian) species. We here identified chicken GRP75 by using immunoprecipitation assay integrated with LC-MS/MS, and found that its amino acid sequence is conserved with high homology (52.5%) to the HSP70 family. Bioinformatics and 3D-structure prediction indicate that, like most HSPs, chicken GRP75 has two principal domains (the NH2-terminal ATPase and COOH-terminal region). Immunofluorescence staining shows that GRP75 is localized predominantly in the avian myoblast and hepatocyte mitochondria. Heat stress exposure upregulates GRP75 expression in a species-, genotype-, and tissue-specific manner. Overexpression of GRP75 reduces avian cell viability, and blockade of GRP75 by its small molecular inhibitor MKT-077 rescues avian cell viability during heat stress. Taken together, this is the first evidence showing that chicken GRP75, unlike its mammalian ortholog, is responsive to heat shock and plays a key role in cell survival/death pathways. Since modern avian species have high metabolic rates and are sensitive to high environmental temperature, GRP75 could open new vistas in mechanistic understanding of heat stress responses and thermotolerance in avian species.