The rate-limiting step in steroidogenesis is the movement of cholesterol from the outer mitochondrial membrane (OMM) to inner mitochondrial membrane (IMM). The steroidogenic acute regulatory protein (StAR) by an unknown mechanism facilitates the movement of cholesterol from the OMM to IMM, thus regulating the flow of substrate to P450scc for steroidogenesis. StAR belongs to a family of proteins called START (StAR related lipid transport domain), where StAR is the primary member. StAR mutations cause a potentially lethal disease called congenital adrenal hyperplasia, in which the synthesis of all steroid hormones is impaired, and the fetuses die shortly after birth. StAR activity is directly proportional to its residency time at the OMM. Our preliminary results show that i) the import mechanism of StAR is unique, ii) the StAR pause sequence has a distinct role in StAR folding and activity, iii) StAR transports more than 100 molecules of cholesterol in one hour, iv) StAR is imported through a complex of several proteins, and v) StAR is imported in a two step process. To understand the mechanism of StAR processing and import into mitochondria, we propose three specific aims.
Aim 1 is to determine the mechanism of activity of newly synthesized StAR. This is critical in understanding how StAR functions with such a short half- life, and how it can reach the OMM with out the N-terminal sequence.
In aim 2, we proposed to determine the mitochondrial site of StAR action, in order to understand the translocation mechanism of StAR by OMM resident proteins VDAC 1 and 2 (Voltage Dependent Anion Channel).
In aim 3, we proposed to determine the unfolding mechanism of StAR by the OMM associated lipid membrane, and to uncover why a specific START-domain is more active than the other. Fulfilling these aims will, I) help us to expand our knowledge about the mechanism of interaction of StAR with the OMM and II) expand our understanding about the role of OMM that help in StAR unfolding and thus mobilization of cholesterol transport. Completing these aims will help us in the understanding of broad aspects of cellular lipid metabolism and also to a broad array of birth defects, disorders of embryogenesis and genetic diseases.

Public Health Relevance

The narrative of this grant application is to determine the mechanism of cholesterol transport into mitochondria by studying the mechanism of import of steroidogenic acute regulatory protein (StAR). StAR requires unfolding at the outer mitochondrial membrane, so the proposed work will identify the interaction of StAR with outer mitochondrial resident protein voltage dependent anion channel (VDAC) and lipids prior to its import. Revealing the StAR import mechanism will help us in the understanding of broad aspects of cellular lipid metabolism and thus, the importance of this project extends far beyond StAR and regulation of steroidogenesis.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Integrative and Clinical Endocrinology and Reproduction Study Section (ICER)
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Yoshinaga, Koji
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Mercer University Macon
Engineering (All Types)
Schools of Engineering
United States
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Prasad, Manoj; Pawlak, Kevin J; Burak, William E et al. (2017) Mitochondrial metabolic regulation by GRP78. Sci Adv 3:e1602038
Prasad, Manoj; Walker, Anna N; Kaur, Jasmeet et al. (2016) Endoplasmic Reticulum Stress Enhances Mitochondrial Metabolic Activity in Mammalian Adrenals and Gonads. Mol Cell Biol 36:3058-3074
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Prasad, Manoj; Kaur, Jasmeet; Pawlak, Kevin J et al. (2015) Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction. J Biol Chem 290:2604-16
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Kaur, Jasmeet; Bose, Himangshu S (2014) Passenger protein determines translocation versus retention in the endoplasmic reticulum for aromatase expression. Mol Pharmacol 85:290-300
Rajapaksha, Maheshinie; Prasad, Manoj; Thomas, James L et al. (2013) Chaperones rejuvenate folding and activity of 3-? hydroxysteroid dehydrogenase 2. ACS Chem Biol 8:1000-8
Rajapaksha, Maheshinie; Kaur, Jasmeet; Bose, Mahuya et al. (2013) Cholesterol-mediated conformational changes in the steroidogenic acute regulatory protein are essential for steroidogenesis. Biochemistry 52:7242-53
Prasad, Manoj; Thomas, James L; Whittal, Randy M et al. (2012) Mitochondrial 3?-hydroxysteroid dehydrogenase enzyme activity requires reversible pH-dependent conformational change at the intermembrane space. J Biol Chem 287:9534-46

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