Proteomic analysis of the mitochondrial glucocorticoid receptor interacting proteins reveals pyruvate dehydrogenase and mitochondrial 60 kDa heat shock protein as potent binding partners

Abstract

Glucocorticoids are steroid hormones that regulate plethora biological actions such as growth and metabolism, immune response, and apoptosis. Glucocorticoids actions are mediated via glucocorticoid receptors which act mainly as transcription factors, but it is also found to be localized in mitochondria. Mitochondrial localization of the receptor indicates novel functions of the receptor. Characterization of the mitochondrial glucocorticoid receptor (mtGR) interacting proteins will shed light on these actions and the biochemical mechanisms that underlie mitochondrial glucocorticoid receptor import and functions. In this study, applying immunoprecipitation, mass spectrometry and Western blot analysis of the GR interacting proteins in total or mitochondrial extracts of HepG2 cells and of HepG2 cells overexpressing a mitochondrial targeted GR we found pyruvate dehydrogenase (PDH), chaperones such as and heat shock protein (HSP) −60, −70, −75 and −90, and 78 kDa glucose-regulated protein, mitochondrial transcription factors and enzymes involved in the regulation of the mitochondrial protein biosynthesis, lipid metabolism, ATP production and apoptosis as glucocorticoid receptor interacting proteins. Our results uncover potential novel mitochondrial partners of the receptor, suggesting possible new regulatory roles of mtGR in the control of mitochondrial-associated functions of the cell. Significance: In this study the mitochondrial GR interacting proteins were characterized highlighting novel regulatory roles of the receptor in mitochondria. Detection of the mtGR/PDH and mtGR/HSP60 interaction in almost all the analyses performed uncovered PDH and HSP60 proteins as potent mtGR binding partners. The interesting finding of the PDH/mtGR interaction possibly indicates involvement of mtGR in the regulation of the balance between glycolytic and oxidative phosphorylation energy production. Characterization of the mitochondrial heat shock −60, −70, −75 and 78 proteins as mtGR binding partners contribute to the characterization of the biochemical mechanisms of the mitochondrial import of the receptor. Moreover, identification of mitochondrial heat shock proteins, metabolic enzymes, transcription factors, OXPHOS, and regulatory molecules in mitochondrial protein biosynthesis as mtGR binding partners indicates possible new regulatory roles of mtGR in the glucocorticoids-induced regulation and orchestration of nuclear and mitochondrial functions, the exact biochemical mechanism of which remain to be established. The study discloses potential new regulatory roles of the receptor in mitochondria, pointing out its importance as a promising target molecule for the control of the mitochondria-associated pathophysiology of the cell. © 2022