An Outer Mitochondrial Translocase, Tom22, Is Crucial for Inner Mitochondrial Steroidogenic Regulation in Adrenal and Gonadal Tissues

After cholesterol is transported into the mitochondria of steroidogenic tissues, the first steroid, pregnenolone, is synthesized in adrenal and gonadal tissues to initiate steroid synthesis by catalyzing the conversion of pregnenolone to progesterone, which is mediated by the inner mitochondrial enzyme 3β-hydroxysteroid dehydrogenase 2 (3βHSD2). We report that the mitochondrial translocase Tom22 is essential for metabolic conversion, as its knockdown by small interfering RNA (siRNA) completely ablated progesterone conversion in both steroidogenic mouse Leydig MA-10 and human adrenal NCI cells. Tom22 forms a 500-kDa complex with mitochondrial proteins associated with 3βHSD2. Although the absence of Tom22 did not inhibit mitochondrial import of cytochrome P450scc (cytochrome P450 side chain cleavage enzyme) and aldosterone synthase, it did inhibit 3βHSD2 expression. Electron microscopy showed that Tom22 is localized at the outer mitochondrial membrane (OMM), while 3βHSD2 is localized at the inner mitochondrial space (IMS), where it interacts through a specific region with Tom22 with its C-terminal amino acids and a small amino acid segment of Tom22 exposed to the IMS. Therefore, Tom22 is a critical regulator of steroidogenesis, and thus, it is essential for mammalian survival.

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Resveratrol Stimulates Cortisol Biosynthesis by Activating SIRT-Dependent Deacetylation of P450scc

Endocrinology ◽

10.1210/en.2011-2088 ◽

2012 ◽

Vol 153 (7) ◽

pp. 3258-3268 ◽

Cited By ~ 18

Author(s):

Donghui Li ◽

Eric B. Dammer ◽

Marion B. Sewer

Keyword(s):

Protein Expression ◽

Fold Increase ◽

Pyridine Nucleotide ◽

Nucleotide Metabolism ◽

Side Chain ◽

Mitochondrial Enzyme ◽

Adrenocortical Cells ◽

Protein Levels ◽

Chain Cleavage ◽

Cleavage Enzyme

In the human adrenal cortex, cortisol is synthesized from cholesterol by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings that activation of ACTH signaling changes the ratio of nicotinamide adenine dinucleotide (NAD) phosphate to reduced NAD phosphate in adrenocortical cells, we hypothesized that pyridine nucleotide metabolism may regulate the activity of the mitochondrial NAD+-dependent sirtuin (SIRT) deacetylases. We show that resveratrol increases the protein expression and half-life of P450 side chain cleavage enzyme (P450scc). The effects of resveratrol on P450scc protein levels and acetylation status are dependent on SIRT3 and SIRT5 expression. Stable overexpression of SIRT3 abrogates the cellular content of acetylated P450scc, concomitant with an increase in P450scc protein expression and cortisol secretion. Mutation of K148 and K149 to alanine stabilizes the expression of P450scc and results in a 1.5-fold increase in pregnenolone biosynthesis. Finally, resveratrol also increases the protein expression of P450 11β, another mitochondrial enzyme required for cortisol biosynthesis. Collectively, this study identifies a role for NAD+-dependent SIRT deacetylase activity in regulating the expression of mitochondrial steroidogenic P450.

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Transcriptional Regulation of Steroidogenic Genes: STARD1, CYP11A1 and HSD3B

Experimental Biology and Medicine ◽

10.3181/0903-mr-97 ◽

2009 ◽

Vol 234 (8) ◽

pp. 880-907 ◽

Cited By ~ 153

Author(s):

Holly A. LaVoie ◽

Steven R. King

Keyword(s):

Transcriptional Regulation ◽

Current Knowledge ◽

Regulatory Protein ◽

Side Chain ◽

Combinatorial Regulation ◽

Cytochrome P450scc ◽

Chain Cleavage ◽

Cleavage Enzyme ◽

Steroidogenic Acute Regulatory ◽

Species Specific

Expression of the genes that mediate the first steps in steroidogenesis, the steroidogenic acute regulatory protein (STARD1), the cholesterol side-chain cleavage enzyme, cytochrome P450scc (CYP11A1) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (HSD3B), is tightly controlled by a battery of transcription factors in the adrenal cortex, the gonads and the placenta. These genes generally respond to the same hormones that stimulate steroid production through common pathways such as cAMP signaling and common actions on their promoters by proteins such as NR5A and GATA family members. However, there are distinct temporal, tissue and species-specific differences in expression between the genes that are defined by combinatorial regulation and unique promoter elements. This review will provide an overview of the hormonal and transcriptional regulation of the STARD1, CYP11A1 and specific steroidogenic HSD3B genes in the adrenal, testis, ovary and placenta and discuss the current knowledge regarding the key transcriptional factors involved.

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The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.15.7247 ◽

1994 ◽

Vol 91 (15) ◽

pp. 7247-7251 ◽

Cited By ~ 77

Author(s):

S. M. Black ◽

J. A. Harikrishna ◽

G. D. Szklarz ◽

W. L. Miller

Keyword(s):

Side Chain ◽

Cytochrome P450scc ◽

Side Chain Cleavage ◽

Chain Cleavage ◽

Cleavage Enzyme

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Homozygous Disruption of P450 Side-Chain Cleavage (CYP11A1) Is Associated with Prematurity, Complete 46,XY Sex Reversal, and Severe Adrenal Failure

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2004-1059 ◽

2005 ◽

Vol 90 (1) ◽

pp. 538-541 ◽

Cited By ~ 75

Author(s):

Olaf Hiort ◽

Paul-Martin Holterhus ◽

Ralf Werner ◽

Christine Marschke ◽

Ute Hoppe ◽

...

Keyword(s):

Molecular Genetic ◽

Laboratory Data ◽

Sex Reversal ◽

Molecular Genetic Analysis ◽

Side Chain ◽

Steroid Synthesis ◽

Cytochrome P450scc ◽

Side Chain Cleavage ◽

Fetal Survival ◽

Chain Cleavage

Abstract Disruption of the P450 side-chain cleavage cytochrome (P450scc) enzyme due to deleterious mutations of the CYP11A1 gene is thought to be incompatible with fetal survival because of impaired progesterone production by the fetoplacental unit. We present a 46,XY patient with a homozygous disruption of CYP11A1. The child was born prematurely with complete sex reversal and severe adrenal insufficiency. Laboratory data showed diminished or absent steroidogenesis in all pathways. Molecular genetic analysis of the CYP11A1 gene revealed a homozygous single nucleotide deletion leading to a premature termination at codon position 288. This mutation will delete highly conserved regions of the P450scc enzyme and thus is predicted to lead to a nonfunctional protein. Both healthy parents were heterozygous for this mutation. Our report demonstrates that severe disruption of P450scc can be compatible with survival in rare instances. Furthermore, defects in this enzyme are inherited in an autosomal-recessive fashion, and heterozygote carriers can be healthy and fertile. The possibility of P450scc-independent pathways of steroid synthesis in addition to the current concept of luteoplacental shift of progesterone synthesis in humans has to be questioned.

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Inner Mitochondrial Translocase Tim50 Is Central in Adrenal and Testicular Steroid Synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.00484-18 ◽

2018 ◽

Vol 39 (1) ◽

Cited By ~ 1

Author(s):

Himangshu S. Bose ◽

Fadi Gebrail ◽

Brendan Marshall ◽

Elizabeth W. Perry ◽

Randy M. Whittal

Keyword(s):

Metabolic Activity ◽

Side Chain ◽

Protein Amino Acid ◽

Steroid Synthesis ◽

Essential Requirement ◽

Chain Cleavage ◽

C Terminus ◽

The Matrix ◽

Cleavage Enzyme ◽

First Time

ABSTRACT Adrenal and gonadal mitochondrial metabolic activity requires electrons from cofactors, cholesterol, and a substrate for rapid steroid synthesis, an essential requirement for mammalian survival. Substrate activity depends on its environment, which is regulated by chaperones and mitochondrial translocases. Cytochrome P450 side-chain cleavage enzyme (SCC or CYP11A1) catalyzes cholesterol to pregnenolone conversion, although its mechanism of action is not well understood. We find that SCC is directly imported into the mitochondrial matrix, where its N-terminal sequence is cleaved sequentially, after which it becomes activated following the second cleavage, which is dependent on the folding of the protein. Following integration of the SCC C terminus into the TIM23 complex, amino acids 141 to 146 interact with the intermembrane-exposed Tim50 protein, forming a large complex. The absence of Tim50 or its mutation reduced enzymatic activity. For the first time, we report that a protein activated at the matrix remains mostly unfolded and is transported back to the IMS to integrate with the TIM23 translocase complex and align with the Tim50 protein. Amino acid changes that suppress the association of Tim50 with SCC ablate metabolic activity. Thus, the TIM23 complex is the central regulator of metabolism guided by Tim50.

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