Characterization of Mitochondrial Heat Shock Protein 60 variants in HEK293 Cells Transformed into Steroidogenic

Abstract

Introduction: During pregnancy, P4 is essential to maintain the maternal-fetal relationship. Maternal cholesterol is the main source of P4 production, a process that takes place in the syncytiotrophoblast mitochondrion. The mechanism and proteins involved in the cholesterol transport for the steroidogenic process are still unknown in detail. The STARD3 protein could be the substitute for its STARD1 equivalent localized in all acute response tissues. However, mutation or null STARD3 mice maintain their reproductive capacity, suggesting other proteins are involved in this process. Previously, we reported that the HSP60 participates in steroidogenesis in mitochondria isolated from the placental syncytiotrophoblast, mitochondrial contact sites or JEG-3. Also, take relevance that non-steroidogenic cells, such as the HEK293, which are human kidney embryo cells, when are transformed into steroidogenic by transfection of the steroidogenic machinery, they synthesize progesterone. To understand better the mechanism through which HSP60 participates in placental steroidogenesis, mutation of cysteine 442, which is essential in the active site for its activity, and deletion of 146 amino acid residues of the N-terminal of HSP60 were performed. The first was implemented to determine whether the protein structure is essential to support steroidogenesis, and the second was done to elucidate whether its activity occurs outside or inside the mitochondrion. Methods: Two mutants were obtained: a) cysteine 442 was replaced by alanine (HSP60C442A) and b) the HSP60-mature (HSP60M) without the mitochondrial-leading sequence. Human kidney cells HEK293 were transformed into steroidogenic by transfection with pECE-P450scc, pCMV-3βHSD-I. The transfected cells were transfected with the HSP60wt, HSP60C442A, or HSP60M plasmids. The transfection was validated by western blot and P4 was determined by an enzyme immunoassay kit. HSP60 without mutations was used as control (HSP60wt). Results: The synthesis of P4 was stimulated by the wild type HSP60 (HSP60wt). However, with both mutants, steroidogenesis occurred as in the control, suggesting that mutants do not support P4 synthesis. Discussion: The mechanism to transport cholesterol to steroidogenic mitochondria requires the full HSP60 to support P4 synthesis, which is necessary to maintain pregnancy. Highlights HSP60 participates in the steroidogenesis of transformed HEK293 cells. Cys442 mutant of HSP60 loses its activity in steroidogenesis. N-terminal deletion of HSP60 is not involved in steroidogenesis. Native HSP60 is critical for steroidogenesis.