The acute regulation of steroid hormone production in steroidogenic tissues is controlled by the delivery of the substrate cholesterol to the inner mitochondrial membrane which is the site of the cholesterol side chain cleavage enzyme system (CSCC). This transfer occurs as a result of the de novo synthesis of a protein(s) whose existence has been known for greater that 30 years but whose identify has remained a mystery until now. While there have been a number of proteins described as being candidates for the acute regulator, unequivocal evidence that any of these proteins performed this function had been lacking. Recently, we purified., cloned and expressed a novel, tropic hormone-induced mitochondrial protein which we had earlier hypothesized to be involved in this regulation. Observations described in this proposal indicate that this protein, which we named the Steroidogenic Acute Regulatory (StAR) protein, is the long sought regulatory protein for acute steroidogenesis. Since we have been successful in characterizing the role of StAR in the acute regulation of steroid production, we would now propose to extend this finding into what can considered to be one of the most obvious directions to take this work, namely, the determination of the mechanism of action whereby StAR is able to effect the transfer of cholesterol from cellular stores and the outer mitochondrial membrane to the inner mitochondrial membrane and the CSCC. Specifically, we propose the following Aims: 1). to characterize the mitochondrial membrane receptor for StAR and determine if it is similar to other mitochondrial protein precursor receptors 2). to determine the cholesterol binding capacity of StAR using filter binding assays as well as steady state polyacrylamide gel assays. 3). to characterize the mitochondrial """"""""contact sites"""""""" in hormone stimulated MA-10 cells and non- stimulated R2C cells in order to determine if StAR and other components of the mitochondrial steroidogenic complex are present at such sites. 4). to construct NH2-terminal mutants for the StAR protein and determine the effects on both mitochondrial import and processing of the protein and on steroidogenesis 5). to construct COOH-terminal deletion mutants for the StAR protein and determine the effects on both mitochondrial import and steroidogenesis. 6). to determine the role of phosphorylation on the activity of StAR by using MA-10 Leydig tumor cells and y 1 adrenal tumor cells. (7). to construct an expression system designed to produce large quantities of the StAR precursor to be used in in vitro systems in order to more specifically demonstrate StAR function and mechanism of action.
These Aims will be performed using standard methodology and their successful completion will add interesting new information concerning the mechanism of action of a novel, hormone induced protein which is specific to steroidogenic tissue. These studies are important since StAR occupies such a fundamental and indispensable role in what can arguably be called one of the most important functions in the body, namely the production of mineralocorticoids, glucocorticoids and gonadal steroids. Indeed, as dramatically pointed out in recent studies, absence of functional StAR results in the condition of lipoid Congenital Adrenal hyperplasia, a lethal syndrome caused by mutations in the StAR gene. In summary, it is our intent to continue to study the characteristics of this novel regulatory protein with the goal of determining the mechanism whereby cholesterol is transferred to the CSCC.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HD017481-15
Application #
6181386
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Yoshinaga, Koji
Project Start
1984-07-01
Project End
2001-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
15
Fiscal Year
2000
Total Cost
$260,537
Indirect Cost
Name
Texas Tech University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Selvaraj, Vimal; Stocco, Douglas M; Clark, Barbara J (2018) Current knowledge on the acute regulation of steroidogenesis. Biol Reprod 99:13-26
Stocco, Douglas M; Zhao, Amy H; Tu, Lan N et al. (2017) A brief history of the search for the protein(s) involved in the acute regulation of steroidogenesis. Mol Cell Endocrinol 441:7-16
Tu, Lan N; Zhao, Amy H; Hussein, Mahmoud et al. (2016) Translocator Protein (TSPO) Affects Mitochondrial Fatty Acid Oxidation in Steroidogenic Cells. Endocrinology 157:1110-21
Selvaraj, Vimal; Stocco, Douglas M; Tu, Lan N (2015) Minireview: translocator protein (TSPO) and steroidogenesis: a reappraisal. Mol Endocrinol 29:490-501
Selvaraj, Vimal; Stocco, Douglas M (2015) The changing landscape in translocator protein (TSPO) function. Trends Endocrinol Metab 26:341-8
Manna, Pulak R; Stetson, Cloyce L; Daugherty, Carol et al. (2015) Up-regulation of steroid biosynthesis by retinoid signaling: Implications for aging. Mech Ageing Dev 150:74-82
Tu, Lan N; Zhao, Amy H; Stocco, Douglas M et al. (2015) PK11195 effect on steroidogenesis is not mediated through the translocator protein (TSPO). Endocrinology 156:1033-9
Stocco, Douglas M (2014) The role of PBR/TSPO in steroid biosynthesis challenged. Endocrinology 155:6-9
Tu, Lan N; Morohaku, Kanako; Manna, Pulak R et al. (2014) Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis. J Biol Chem 289:27444-54
Manna, Pulak R; Slominski, Andrzej T; King, Steven R et al. (2014) Synergistic activation of steroidogenic acute regulatory protein expression and steroid biosynthesis by retinoids: involvement of cAMP/PKA signaling. Endocrinology 155:576-91

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