Steroid hormones are ubiquitous regulators of physiologic processes and are essential for life. The human body makes a broad array of steroid hormones in numerous tissues but the biosynthesis of all steroid hormones begins with cholesterol. Work over the last 15-20 years has delineated the pathways of steroidogenesis, leading to the identification and characterization of all the enzymes of steroidogenesis and the cloning of their genes. Our previous work supported by DK37922 included the cloning and characterization of many of these cDNAs and genes and has emphasized the delineation of the biological role of each of these by studying the human genetic diseases that result from their mutations. A major focus of our work for the past 6 years has been the steroidogenic acute regulatory protein (StAR). StAR facilitates the movement of cholesterol from the outer to inner mitochondrial membrane, and we showed that StAR mutations cause congenital lipoid adrenal hyperplasia; however the mechanism of StAR's action remains unknown and controversial. To elucidate the mechanisms of StAR's action we propose five Aims.
Aim 1 : Determine the mitochondrial site of StAR's action. Our data indicate that StAR acts on the cytoplasmic side of the outer mitochondrial membrane (0MM), but others have suggested that StAR functions between the outer and inner membranes, in the intra-membranous space.
Aim 2 : Determine whether the mechanisms of StAR's import into the mitochondria influence StAR's activity. If StAR acts on the 0MM, its net activity should be related to 0MM residency time, and mitochondrial import should inactivate StAR.
Aim 3 : Determine whether StAR undergoes conformational changes while interacting with membrane models of the 0MM. We have reported that StAR undergoes a pH-dependent change to a molten globule conformation in solution, and in association with a synthetic membrane, but it is not yet known if this happens in association with the 0MM.
Aim 4 : Determine whether StAR undergoes conformational changes while binding and discharging cholesterol. Understanding how StAR associates with and discharges cholesterol is crucial for understanding how StAR moves cholesterol from the outer to inner mitochondrial membrane.
Aim 5 : Identify the domains (sequences) of N-62 StAR that interact with the outer mitochondrial membrane. The topology of StAR's interactions with the mitochondrion have not yet been explored in any published work;
this aim will determine which segments of the StAR protein associate with the 0MM. Fulfilling these 5 aims will substantially advance our understanding of the mechanism of StAR's action.
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