Mitochondrial endocrinology Mitochondria as key to hormones and metabolism
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). StAR and StarD6 stimulate the movement of cholesterol from the OMM to the inner mitochondrial membrane (IMM), but StarD4 and StarD5 do not ( Bose et al., 2008a ). StarD4 and/or StarD5 may bring cholesterol to the OMM; however, StarD4 knockout mice have no changes in steroidogenesis and minimal changes in weight and serum lipids, hence an essential function of StarD4 is not established ( Riegelhaupt et al., 2010 ). While all the mecha- nisms that move cholesterol to the mitochondria have not been determined, the current view is that the principal mechanism is by non-vesicular transport via StarD proteins, but that StAR itself plays a minor role in this step. The OMM of adrenal mitochondria contains abundant cholesterol, while the IMM contains little cho- lesterol. Whether or not all OMM cholesterol is potentially avail- able for steroidogenesis has been unclear. Early studies identified a distinct pool of ‘‘steroidogenic’’ OMM cholesterol that was dis- tinct from the structural membrane cholesterol and could be mobi- lized by cAMP ( Stevens et al., 1992 ). Movement of cholesterol from the OMM to the IMM is central to the initiation of steroidogenesis, because the cholesterol side-chain cleavage enzyme (P450scc) sys- tem is localized to the IMM. Whether or nor there are two kineti- cally distinct pools of cholesterol in the OMM, and how cholesterol is transferred to the IMM remain under investigation. 3. Entry of cholesterol into steroidogenic mitochondria: action of the steroidogenic acute regulatory protein, StAR 3.1. Acute and chronic regulation of steroidogenesis Unlike cells that produce polypeptide hormones, which store large amounts of mature hormone available for rapid release, cells that produce steroid homones store very little steroid, so that ste- roid secretion requires induction of steroid synthesis. There are several distinct mechanisms regulating steroidogenesis. In the adrenal, ACTH promotes steroidogenesis at three levels. First, over the course of months ACTH promotes adrenal growth via fibroblast growth factor, epidermal growth factor, insulin-like growth factor 2, and possibly other factors. Second, over the course of days, ACTH, acting via cAMP in the adrenal zona fasciculata and angio- tensin II acting via calcium/calmodulin in the zona reticularis pro- mote transcription of genes for steroidogenic enzymes, especially the gene encoding P450scc (CYP11A1) thus increasing the amounts of steroidogenic enzymes. Third, acting on a 15-60 min time scale, ACTH rapidly stimulates both the activation of pre-existing StAR and the synthesis of new StAR. ACTH/cAMP stimulates phosphory- lation of StAR at Ser195 almost immediately, doubling its activity ( Arakane et al., 1997 ), and induces StAR transcription within min- utes (
Jo et al., 2005; Manna et al., 2009 ). StAR then interacts with a complex macromolecular machine on the OMM to increase the flow of cholesterol from the OMM to the IMM where it becomes the substrate for P450scc. The first two modes of steroid regulation comprise the chronic steroidogenic response and the action of StAR comprises the acute steroidogenic response. 3.2. The steroidogenic acute regulatory protein, StAR StAR was discovered in the search for the factor that triggers the acute steroidogenic response. Autoradiographic spots were noted on 2-dimentional gels that appeared within 30 min of stimulating steroidogenic tissues ( Pon and Orme-Johnson, 1986; Pon et al., 1986; Epstein and Orme-Johnson, 1991; Stocco and Sodeman 64 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 1991 ); the cDNA was then cloned ( Clark et al., 1994; Sugawara et al., 1995 ), and the factor was named the steroidogenic acute reg- ulatory protein, StAR (reviewed in Stocco and Clark, 1996 ). StAR is a 37-kDa protein with an N-terminal mitochondrial targeting se- quence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. Co-expression of StAR and P450scc in nonsteroidogenic cells rapidly increases conversion of cholesterol to pregnenolone, suggesting that StAR triggers the acute steroidogenic response ( Stocco and Clark, 1996 ). The indis- pensible role of StAR was demonstrated by finding that StAR muta- tions cause congenital lipoid adrenal hyperplasia, in which very little steroid is made ( Lin et al., 1995; Bose et al., 1996 ). However, some steroidogenesis can take place in the absence of StAR. In vivo, the human placenta synthesizes steroids via mitochondrial P450scc but does not express StAR ( Bose et al, 2000 ). In vitro, cells expressing the P450scc enzyme system but not StAR can convert cholesterol to pregnenolone ( Harikrishna et al., 1993; Black et al., 1994
) at 14% of the maximal StAR-induced rate ( Lin et al., 1995; Bose et al., 1996 ). StAR-independent steroidogenesis might occur without a triggering protein, possibly using intracellular hydroxysterols that bypass StAR action; alternatively, another pro- tein, such as the 30 kDa proteolytic cleavage product of MLN64, may exert StAR-like activity to promote mitochondrial cholesterol import, but the precise mechanism(s) are unclear. 3.3. StAR acts on the outer mitochondrial membrane The presence of a typical mitochondrial ‘leader’ sequence ini- tially suggested that 37 kDa cytoplasmic StAR is a ‘precursor. and that the 30 kDa intramitochondrial protein is the ‘mature form’, but these terms do not describe the biology of StAR ( Miller, 2007 ). The crystallographic structure of the START domain of MLN64 (N- 216 MLN64), which shares 37% amino acid sequence identity with StAR, suggested that 30-kDa StAR acts in the intramembranous space (IMS) to shuttle cholesterol from the OMM to the IMM ( Tsuj-
ishita and Hurley, 2000 ). This structure shows a globular protein with an a
approximately 26 Å deep and 10 Å across at its widest diameter. The structure of StAR was then modeled based on MLN64 ( Mathieu et al., 2002; Yaworsky et al., 2005 ) and these models have been con- firmed by crystallography at 3.4 Å resolution ( Thorsell et al., 2011 ). The sterol-binding pocket of StAR accommodates a single molecule of cholesterol with its 3b-OH group coordinated by the two polar residues. These structures and the structure of StarD4 ( Romanowski et al., 2002 ) are characterized by long N- and C-terminal a -helixes, two short a -helixes, and 9 antiparallel b sheets that form a helix- grip fold. However, deletion of StAR’s mitochondrial leader has no effect on its activity ( Arakane et al., 1996 ), and both the 37 kDa and 30 kDa forms of StAR are equally active in vivo and in vitro ( Bose
et al., 2002 ). When StAR was fused to the cytoplasmic C-terminus of the OMM protein Tom-20, thus immobilizing StAR an OMM, it was constituitively active, but when StAR was immobilized in the IMS or on the matrix side of the IMM, it was wholly inactive. Furthermore, manipulating the speed of StAR’s mitochondrial entry by manipu- lating the leader sequence showed that faster import decreased activity whereas slower import increased activity ( Bose et al., 2002
). Thus, StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. Thus it is the OMM localization of StAR, and not its cleavage from the 37 kDa form to the 30 kDa form, that determines its activity. 3.4. StAR’s action requires conformational changes and serine phosphorylation The mechanism by which StAR triggers cholesterol flux from the OMM to the IMM involves a multi-protein complex on the OMM (see below) and requires pH-induced conformational changes in StAR that are needed for StAR to accept and discharge cholesterol ( Bose et al., 1999; Baker et al., 2005 ). StAR can transfer cholesterol between synthetic, protein-free membranes in vitro, but with non-physiologic stoichiometry ( Tuckey et al., 2002 ), and a biologically inactive StAR mutant (R182L) can also transfer cho- lesterol to membranes in vitro ( Baker et al., 2007 ). Thus choles- terol-binding is necessary but not sufficient for StAR activity. Proteolysis of StAR bound to synthetic membranes that model OMM lipid composition showed that only the exterior surface of the C-terminal a -helix and small segments of the adjacent X -loops
were protected, suggesting that only these domains interact with the OMM ( Yaworsky et al., 2005 ). Molecular dynamics studies show that cholesterol is blocked from reaching the cholesterol- binding pocket of StAR by a set of hydrogen bonds that are dis- rupted when the surface residues of StAR are protonated (as hap- pens when StAR interacts with charged phospholipids on the OMM), thus eliciting a conformational change that permits choles- terol access ( Bose et al., 1999; Baker et al., 2005 ). When the move- ment of the C-helix is prohibited by mutagenesis that creates disulfide bonds, activity is lost, but reducing these bonds restores activity, showing that the immobilized C-helix, and not the muta- genesis, is responsible for the lost activity ( Baker et al., 2005 ). Thus
the activity of StAR on the OMM requires an acid-induced disrup- tion of hydrogen bonds and a consequent conformational change in StAR to permit it to bind and release cholesterol. The phosphorylation of StAR on Ser 195 approximately doubles StAR’s activity ( Arakane et al., 1997 ). It appars that the protein ki- nase A (PKA) anchor protein AKAP121 recruits the type II PKA reg- ulatory subunit a (PKAR2A) to the OMM, which phosphorylates StAR, whereas the type I kinase drives StAR transcription ( Dyson
et al., 2009 ). A physical interaction between the 37 kDa cytoplas- mic form of StAR and HSL has also been reported ( Shen et al., 2003 ). StAR is recycled, with each molecule moving hundreds of molecules of cholesterol into the mitochondria before StAR is im- ported into the mitochondria and is inactivated ( Artemenko et al., 2001 ). 4. StAR interacts with an OMM protein complex 4.1. The peripheral benzodiazepine receptor or mitochondrial transporter protein StAR acts on the OMM via a complex that consists of several proteins, most of which are now identified, even though it remains unclear what role each plays in the mitochondrial importation of cholesterol ( Bose et al., 2008b; Rone et al., 2012; Papadopoulos and Miller, 2012 ). The first of these proteins to be identified is the peripheral benzodiazepine receptor (PBR) ( Lacapere and Papadopoulos, 2003 ), now also called the mitochondrial trans- porter protein (TSPO) ( Papadopoulos et al., 2006 ). It was initially proposed that PBR/TSPO was the ‘acute trigger’ of steroidogenesis, but it is now clear that StAR plays that role, and that PBR/TSPO is part of the molecular machine on the OMM through which StAR acts (
Papadopoulos and Miller, 2012 ). PBR/TSPO is a ubiquitously expressed 18 kDa protein that comprises up to 2% of OMM protein. PBR/TSPO ligands can stimulate cholesterol movement from the OMM to the IMM and stimulate steroidogenesis ( Lacapere and Papadopoulos, 2003 ). Knockdown of the gene encoding PBR/TSPO in Leydig cells disrupts cholesterol transport and steroidogenesis, and transfection of the PBR/TSPO-disrupted cells with the wild- type cDNA rescues steroidogenesis ( Papadopoulos et al., 1997 ). However, knocking out the PBR/TSPO gene in mice caused early embryonic lethality, indicating its indispensible role during devel- opment (
Lacapere and Papadopoulos, 2003 ). W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 65 As is typical of proteins associated with the OMM, PBR/TSPO lacks a cannonical mitochondrial targeting sequence; instead, the C-terminal half targets PBR/TSPO to the OMM ( Rone et al., 2009 ). PBR/TSPO appears to have five transmembrane a -helices spanning the OMM ( Joseph-Liauzun et al., 1998; Korkhov et al., 2010 ), sug- gesting that TSPO functions as a cholesterol channel, consistent with data indicating that PBR/TSPO acts downstream from StAR ( Hauet et al., 2005; Liu et al., 2006 ). PBR/TSPO and has a cytoplas- mic domain containing a ‘‘cholesterol recognition amino acid con- sensus’’ (CRAC) domain ( Li et al., 2001 ). PBR/TSPO binds cholesterol at the CRAC domain, suggesting that this domain participates in transferring cholesterol from the OMM to the IMM ( Jamin et al., 2005 ). Mutagenesis of the CRAC domain interferes with cholesterol binding and transfer of cholesterol to the IMM, and blocking the binding of cholesterol to the CRAC domain prevents steroidogene- sis ( Midzak et al., 2011 ). Targeted deletion of the TSPO gene in an immortalized Leydig cell line blocked cholesterol transport into the mitochondria and reduced steroid production; reintroduction of TSPO into the deficient cell line restored steroidogenic capacity ( Li et al., 2001 ). 4.2. Other components of the OMM protein complex PBR/TSPO is a component of a 140–200 kDa multi-protein com- plex consisting of PBR/TSPO, the 34-kDa voltage-dependent anion channel (VDAC1), the 30-kDa the adenine nucleotide transporter (ANT), the 10-kDa diazepam-binding inhibitor (acyl-CoA-binding domain 1, ACBD1), the TSPO-associated protein-1 (PRAX-1), and the PKA regulatory subunit RI a -associated protein 7 (PAP7) ( Rone et al., 2012 ). PAP7, also known as acyl-CoA binding domain-con- taining protein 3 (ACBD3), binds both TSPO and the regulatory sub- unit RI a
Fan et al., 2010 ). PBR/TSPO interacts with VDAC on the OMM, helping to anchor the multi-protein complex to the OMM and assisting with the binding and import of StAR ( Liu et al., 2006 ); VDAC1 also interacts with ANT on the IMM ( McEnery
et al., 1992 ). While a functional interaction between StAR and PBR/ TSPO is clear ( Hauet et al., 2005 ), a physical interaction has not been demonstrated; protein cross-linking experiments indicate that StAR directly interacts with VDAC1 and with phosphate carrier protein (PCP), but not with PBR/TSPO ( Bose et al., 2008b ). VDAC1
also appears to interact with PBR/TSPO ( Liu et al., 2006; Rone et al., 2009 ). It is not clear how VDAC-1 participates in cholesterol import, as VDAC-1 has a ring-like structure with a hydrophilic inte- rior that is well-suited to anion transport, but is unlikely to form a channel for hydrophobic cholesterol ( Bayrhuber et al., 2008; Hiller et al., 2008 ). VDAC is found at contact sites between the OMM and the IMM ( Mannella et al., 1992 ) where it may complex with hexo- kinase, ANT, creatine kinase and proteins of the Bcl-2 family ( Brdiczka et al., 2006 ). 5. Mutations in StAR – congenital lipoid adrenal hyperplasia (Lipoid CAH) Lipoid CAH is characterized by absent or very low serum con- centrations of all steroids, high basal ACTH and plasma renin activ- ity, and grossly enlarged adrenals filled with cholesterol and cholesteryl esters ( Miller, 1997 ). Lipoid CAH was initially thought to be an enzymatic disorder and was mis-termed ‘20,22-desmolase deficiency’, but the CYP11A1 gene for P450scc is not mutated in these patients ( Lin et al., 1991 ), and the placenta (a fetal tissue) continues to produce progesterone in lipoid CAH ( Saenger et al., 1995 ), indicating a lesion upstream from P450scc, such as in a fac- tor involved in mitochondrial cholesterol transport ( Lin et al., 1991 ). Mutations in the gene for PBR/TSPO were sought and ex- cluded ( Lin et al., 1993 ), but mutations were found in StAR ( Lin
et al., 1995; Bose et al., 1996 ), so that lipoid CAH represents the StAR knockout experiment of nature ( Miller, 1997 ). The pathophysiology of lipoid CAH is explained by the two-hit model ( Bose et al., 1996 ). The first hit is lost StAR activity, causing diminished steroidogenesis and a compensatory rise in ACTH and gonadotropins. These increased tropic hormones stimulate intra- cellular cAMP production, increasing LDL receptors, LDL choles- terol uptake, and de novo synthesis of cholesterol. Intracellular cholesterol then accumulates, causing the second hit: the loss of remaining steroidogenic capacity due to mitochondrial damage from the accumulated cholesterol, cholesteryl esters, and their auto-oxidation products ( Bose et al., 1996 ) ( Fig. 2
). The absence of StAR activity affects fetal testicular Leydig cells early in gesta- tion, preventing synthesis of the testosterone needed for the devel- opment of male external genitalia, hence affected 46,XY fetuses are born with female external genitalia. However, the testicular Sertoli cells function normally and produce anti-Müllerian hormone, so the phenotypically female 46,XY fetus lacks female internal repro- ductive organs. The fetal adrenal typically produces very little aldosterone, so that the zona glomerulosa is not tropicly stimu- lated and hence usually remains undamaged until after birth. Hence, newborns with lipoid CAH may not have a salt-wasting cri- sis until several months of life, when chronic stimulation then leads to cellular damage. The two-hit model has been confirmed in clinical studies, explaining why affected 46,XX patients feminize at the age of pub- erty (
Bose et al., 1997; Fujieda et al., 1997 ), and also in knockout mice ( Hasegawa et al., 2000 ). The fetal ovary makes essentially no steroids and remains unstimulated and hence undamaged until adolescence, when it is stimulated by pubertal gonadotropins, yielding small amounts of estrogen produced by StAR-independent steroidogenesis. However, this low level of steroidogenesis is insuf- ficient to generate progesterone in response to the mid-cycle LH surge; continued stimulation results in cholesterol accumulation and cellular damage, impairing the later biosynthesis of progester- one, resulting in anovulatory cycles. Gonadotropin stimulation does not promote global ovarian steroidogenesis, but only recruits individual follicles, hence most follicles remain undamaged and available for future cycles. A new follicle is recruited with each suc- cessive cycle, producing estradiol by StAR-independent steroido- genesis, resulting in breast development, so that the patient experiences general feminization, monthly estrogen withdrawal and cyclic vaginal bleeding. Lipoid CAH is relatively common in Japan and Korea, where the carrier frequency is approximately one in 300, and most affected individuals carry the mutation Q258X ( Bose et al., 1996; Nakae et al., 1997; Kim et al., 2011 ). Mutations retaining about 10–25% of normal StAR activity, especially R188C, cause a milder disease called ‘‘non-classic lipoid CAH’’, characterized by adrenal insuffi- ciency several years after infancy, nearly normal masculinization of 46,XY individuals, and minimally affected mineralocorticoid secretion ( Baker et al., 2006 ). 6. The cholesterol side chain cleavage enzyme – the prototypical mitochondrial P450 6.1. Cytochrome P450 enzymes Once cholesterol reaches the IMM, it may be converted to preg- nenolone to initiate steroidogenesis. Most steroidogenic enzymes are cytochrome P450 enzymes, all of which have approximately 500 residues, contain a single heme group and absorb light at 450 nm when reduced with carbon monoxide. The human genome contains 57 CYP genes encoding cytochrome P450 enzymes; the corresponding proteins may be given the same name without the 66 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 use of italics (thus CYP11A1 encodes the cholesterol side chain cleavage enzyme, P450scc; which may also be termed CYP11A1, without italics). Seven human cytochrome P450 enzymes are tar- geted to the mitochondria, the other 50 are targeted to the ER; the roles of the human P450 enzymes have been reviewed recently ( Nebert et al., 2013 ). P450 enzymes activate molecular oxygen via their heme iron using electrons donated by nicotinamide adenine dinucleotide phosphate (NADPH). Mitochondrial (type 1) P450 en- zymes receive electrons from NADPH via an electron transfer chain consisting of a flavoprotein termed ferredoxin reductase and a small iron-sulfur protein termed ferredoxin ( Miller, 2005 ) ( Fig. 3 ). The type 2 P450 enzymes in the ER receive electrons via a single 2-flavin protein termed P450 oxidoreductase ( Miller,
2005 ). All P450 enzymes can catalyze multiple chemical reactions, often with very different substrates. 6.2. Steps in steroidogenesis Download 2.44 Mb. Do'stlaringiz bilan baham: 
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