Mitochondrial endocrinology Mitochondria as key to hormones and metabolism
participate in steroidogenesis, and at least
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Six P450 enzymes participate in steroidogenesis, and at least three more participate in the processing of vitamin D; five of these are found in mitochondria. Steroidogenesis is initiated by mito- chondrial P450scc. P450scc cleaves the 20,22 bond of insoluble cholesterol to produce soluble pregnenolone, and is the hormon- ally regulated, rate-limiting step in steroidogenesis. It is the expression of the CYP11A1 gene that renders a cell ‘steroidogenic’. Pregnenolone may then be converted to progesterone by 3b- hydroxysteroid dehydrogenase (3bHSD), which may be found both in the mitochondria and in the ER (see Section 9). Alternatively, pregnenolone may exit the mitochondrion and become the sub- strate for P450c17 in the ER, which catalyzes both 17 a -hydroxy- lase and 17,20 lyase activities. Pregnenolone appears to exit the mitochondrion unaided; no transport protein has been found, and physiologic evidence does not suggest the presence of such a transporter. Following the activities of 3bHSD and P450c17, Fig. 2. Two-hit model of lipoid CAH. (A) In normal adrenal cells, cholesterol is primarily derived from low-density lipoproteins, and the rate-limiting step in steroidogenesis is movement of cholesterol from the OMM to the IMM. (B) Early in lipoid CAH, StAR-independent steroidogenesis moves small amounts of cholesterol into mitochondria, yielding sub-normal steroidogenesis; ACTH secretion increases, stimulating further accumulation of cholesteryl esters in lipid droplets. (C) As lipids accumulate, they damage the cell through physical engorgement and by the action of cholesterol auto-oxidation products; steroidogenic capacity is destroyed, but tropic stimulation continues. Ovarian follicular cells remain unstimulated and undamaged until puberty, when small amounts of estradiol are produced, as in B, causing phenotypic feminization, with infertility and hypergonadotropic hypogonadism. Modified from ( Bose et al., 1996 ), with permission. Fig. 3. Organization of mitochondrial P450 enzyme systems. NADPH first donates electrons to the FAD moiety of ferredoxin reductase (FeRed); ferredoxin reductase then interacts with ferredoxin (Fedx) by charge-charge attraction, permitting electron transfer of the Fedx to the Fe 2 S
center (ball and stick diagram). Ferredoxin then dissociates from ferredoxin reductase and diffuses through the mitochrondrial matrix. The same surface of ferredoxin that received the electrons from ferredoxin reductase then interacts with the redox-partner binding-site of a mitochondrial P450, such as P450scc, and the electrons then travel to the heme ring of the P450. The heme iron then mediates catalysis with substrate bound to the P450. ÓWL Miller.
W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 67
P450c21 catalyzes the 21-hydroxylation of both glucocorticoids and mineralocorticoids. The final steps in the synthesis of both glu- cocorticoids and mineralocorticoids again takes place in the mito- chondria, where two proteins that share 93% sequence identity, 11b-hydroxylase (P450c11b, CYP11B1), and aldosterone synthase (P450c11AS, CYP11B2) reside. P450c11b catalyzes the 11b-hydrox- ylation of 11-deoxycortisol to cortisol, and P450c11AS catalyzes the 11b-hydroxylation, 18-hydroxylation, and 18-methyl oxidation to convert deoxycorticosterone to aldosterone. Histologic and elec- tron microscopic examination of steroidogenic cells suggests that domains of the ER containing the steroidogenic P450 enzymes come close to the OMM during hormonally-induced steroidogene- sis, forming a steroidogenic complex, so that the movement of ste- roidal intermediates from the mitochondrion to the ER involves very small distances. 6.3. Chemistry and transcription of P450scc P450scc catalyzes three reactions: 22-hydroxylation of choles- terol, 20-hydroxylation of 22(R)-hydroxycholesterol, and oxidative scission of the C20-22 bond of 20(R),22(R)-dihydroxycholesterol, yielding pregnenolone and isocaproaldehyde. The binding of cho- lesterol and 22-hydroxylation are rate-limiting, as the efficiencies (k cat /Km) are much higher for the subsequent reactions, and the high K
D of 3000 nM drives the dissociation of pregnenolone from P450scc ( Miller and Auchus, 2011 ). Alternatively, soluble hydrox- ysterols such as 22(R)-hydroxycholesterol can enter the mitochon- drion readily, without the action of StAR and its associated machinery. Catalysis by P450scc is slow, with a net turnover of approximately 6–20 molecules of cholesterol per molecule of P450scc per second. The crystal structures of bovine ( Mast et al., 2011
) and human ( Strushkevich et al., 2011 ) P450scc, the latter in complex with ferredoxin, show that the single active site of P450scc is in contact with the IMM. Transcription of the CYP11A1 gene determines cellular steroido- genic capacity. This transcription is regulated by hormonally- responsive factors that differ in different types of steroidogenic cells; both the PKA and PKC second messenger systems can induce CYP11A1
transcription via different promoter elements. Adrenal and gonadal transcription of P450scc and other steroidogenic en- zymes requires the action of steroidogenic factor 1 (SF1) ( Schim-
mer and White, 2010 ). By contrast, placental expression of P450scc is constitutive, independent of SF1 and requires members of the CP2 (graineyhead) family of transcription factors (also known as LBP proteins) ( Huang and Miller, 2000; Henderson et al., 2007 ) and TreP-132 ( Gizard et al., 2002 ). Thus, long-term cellular stimu- lation over the course of days will increase the content of P450scc, and the level of basal steroid produced, as well as the capacity of the cell to mount a steroidogenic response. 7. Electron transfer to P450scc: ferredoxin reductase and ferredoxin 7.1. Ferredoxin reductase Catalysis by P450scc and other mitochondrial P450 enzymes re- quires two electron-transfer intermediates, ferredoxin reductase and ferredoxin ( Miller, 2005 ). Ferredoxin reductase receives elec- trons from NADPH then forms a 1:1 complex with ferredoxin, which then dissociates and forms an analogous 1:1 complex with a mitochondrial P450 such as P450scc, thus functioning as an indiscriminate, diffusible electron shuttle for all mitochondrial forms of P450 ( Fig. 3 ). The relative abundances of ferredoxin reduc- tase and ferredoxin, as well as the inherent properties of the mito- chondrial P450, determine catalytic activity ( Harikrishna et al., 1993
). Genetic disorders of human ferredoxin reductase and ferre- doxin have not been described, and mouse knockouts have not been reported. Mutation of the Drosophila ferredoxin reductase homologue dare causes developmental arrest and degeneration of the adult nervous system secondary to disrupted ecdysone produc- tion (
Freeman et al., 1999 ). The human FDXR gene produces two alternatively spliced ferre- doxin reductase mRNAs differing by 18 bp ( Solish et al., 1988 ), but only the protein encoded by the more abundant, shorter mRNA is active ( Brandt and Vickery, 1992 ). Ferredoxin reductase mRNA is widely expressed, but is far more abundant in steroidogenic tissues ( Brentano et al, 1992 ). Ferredoxin reductase is a 54.5 kDa flavopro- tein affixed to the IMM that consists of two domains, each com- prising a b-sheet core surrounded by a -helices ( Ziegler et al., 1999
). The NADP(H)-binding domain is compact, whereas the do- main that binds flavin adenine dinucleotide (FAD) is more open; this domain binds the dinucleotide portion of FAD across a Ross- man fold with the redox-active flavin isoalloxazine ring abutting the NADP(H) domain. Electron transfer occurs in the cleft formed by these two domains. This cleft is characterized by basic residues that interact with acidic residues on ferredoxin. 7.2. Ferredoxin The human FDX1 gene encodes ferredoxin (Fdx1), a 14 kDa, sol- uble, iron/sulfur (Fe 2 S
) protein that resides either free in the mito- chondrial matrix or is loosely bound to the inner mitochondrial membrane ( Miller, 2005 ). There are two human ferredoxins, Fdx1 and Fdx2, but only Fdx1 supports steroidogenic mitochondrial P450 enzymes; Fdx2 participates in the synthesis of heme and Fe/S cluster proteins ( Sheftel et al., 2010 ). Ferredoxin has a core re- gion containing four cysteine residues that tether the Fe 2 S 2 cluster,
and an interaction domain containing a helix with several charged residues, producing a negatively charged surface above the Fe 2 S
cluster that interacts with the mitochondrial P450 ( Muller et al., 1998 ).
The same surface of ferredoxin interacts with both ferredoxin reductase and the P450; nevertheless, creation of three-component fusion proteins of the general scheme H 2 N-P450-FerredoxinReduc- tase-Ferredoxin-COOH (termed F2) increases Vmax. This design was first tested with P450scc ( Harikrishna et al., 1993 ) and has been confirmed with P450c27 ( Dilworth et al., 1996 ) and P450c11b ( Cao et al., 2000 ). In such fusions, the ferredoxin moiety is at the C-terminus, tethered by a hydrophilic linker that permits rotational freedom, so that the same surface of the ferredoxin can access both the P450 and the ferredoxin reductase. The require- ment for ferredoxin reductase and ferredoxin is not absolute, at least in vitro. When P450scc is fused to an alternative electron do- nor, microsomal P450 oxidoreductase and targeted of to the mito- chondria, it remains active; by contrast when P450scc, its fusion protein, or other P450scc constructs are targeted to the ER, they are inactive even when supplied with the 22(R)-hydroxycholes- terol substrate that bypasses the StAR system ( Black et al., 1994 ). Thus the mitochondrial localiztion is essential for the enzymatic activity of P450scc. 8. P450scc deficiency syndromes Three models of defective P450scc function, a spontaneously occurring CYP11A1 deletion in the rabbit ( Yang et al., 1993 ), knock- out of the gene in the mouse ( Hu et al., 2002 ), and rare patients with P450scc mutations confirm that P450scc is the only enzyme 68 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 that converts cholesterol to pregnenolone. Because progesterone is needed to suppress uterine contractility and thus prevent sponta- neous abortion, it would appear that P450scc mutations would be incompatible with term gestation; the mouse and rabbit models are explained by the persistence of the maternal corpus luteum in these species, providing an alternative source of progesterone. Nevertheless, beginning in 2001 ( Tajima et al., 2001 ) 19 patients have been described with mutations in CYP11A1 that affect P450scc activity ( Tee et al., 2013 ). Most of these patients have mutations that ablate all P450scc activity; they probably reached term gestation because of the maternal corpus luteum remained functional beyond the second trimester, when it normally invo- lutes. These patients may be clinically indistinguishable from those with lipoid CAH. The 46,XY genetic males fail to produce testoster- one during fetal life, and are born with female external genitalia, although their internal reproductive structures are male, as their testes produced anti-Müllerian hormone. Following birth, these patients require steroid hormone replacement therapy and may have long-term survival. As with non-classical lipoid CAH, a milder ‘‘non-classical’’ form of P450scc deficiency has been described caused by missense mutations that retain 10–20% of normal activ- ity (
Rubtsov et al., 2009; Sahakitrungruang et al., 2011 ). No hor- monal test distinguishes lipoid CAH from P450scc deficiency, but the adrenals are typically grossly enlarged in lipoid CAH but nor- mal-sized in P450scc deficiency, sometimes permitting radiologic distinction, but the only definitive test to distinguish these disor- ders is DNA sequencing ( Gucev et al., 2013 ). 9. 3b-hydroxysteroid dehydrogenase The 42 kDa 3b-hydroxysteroid dehydrogenase (3bHSD) is a member of the short-chain dehydrogenase/reductase (SDR) family of enzymes, which are are b- a -b proteins having up to seven par- allel b-strands that fan across the center of the molecule, forming the so-called ‘‘Rossman fold’’, which is characteristic of oxida- tion/reduction enzymes that use nicotinamide cofactors ( Agarwal
and Auchus, 2005; Penning, 1997 ). 3bHSD converts D 5 steroids (pregnenolone, 17OH-pregneneolone, DHEA), having a double bond in the B ring to D 4 steroids, having a double bond in the A ring ( Miller and Auchus, 2011 ). Members of this family of enzymes lack mitochondrial leader peptides and are generally found in the cytosol. However, 3bHSD was first isolated from mitochondria ( Thomas et al., 1989 ). This unexpected cellular localization was confirmed by immunogold electron microscopy showing that 3bHSD immunoreactivity is found in mitochondria and endoplas- mic reticulum as well as in the cytoplasm of bovine adrenal zona glomerulosa cells ( Cherradi et al., 1997; Pelletier et al., 2001 ). It is not clear if this is also true for human 3bHSD, or if this subcellu- lar distribution differs in various types of steroidogenic cells, but this property could be a novel mechanism for regulating the direc- tion of steroidogenesis. 3bHSD appears to be associated with P450scc on the IMM, and possibly at OMM-IMM contact sites ( Cherradi et al., 1995 ). 3bHSD and 17 a -hydroxylase (P450c17) compete for the pregnenolone produced by P450scc. The Michaelis constant (Km) for P450c17 in the ER is about 0.8 l M (
Auchus et al., 1998
), whereas the Km of 3bHSD is about 5.2–5.5 l M ( Lee et al., 1999
), so that an intramitochondrial location of 3bHSD will facili- tate the formation of progesterone rather than 17OH-pregneno- lone. During its mitochondrial entry, 3bHSD associates with several mitochondrial translocase proteins to reach the IMM ( Paw- lak et al., 2011 ) and undergoes a pH-dependent conformational change in the intramembranous space that facilitates its two enzy- matic activities ( Prasad et al., 2012 ). There are two 3bHSD genes and several pseudogenes in a gene cluster on chromosome 1p13.1. The two encoded 3bHSD enzymes are 93.5% identical with nearly indistinguishable enzymology, but with distinct distributions of expression. The type 1 enzyme cata- lyzes 3bHSD activity in placenta, breast, liver, brain and some other tissues, whereas the type 2 enzyme is expressd in the adrenals and gonads. Deficiency of 3bHSD2 causes a rare form of congenital adrenal hyperplasia (3bHSD deficiency); mutations have not been found in 3bHSD1, possibly reflecting the impact of such a mutation on plcental progesterone synthesis. 10. Other steroidogenic mitochondrial P450 enzymes Adrenocortical mitochondria contain two additional P450 en- zymes: P450c11b (11b-hydroxylase) is found in zona fasciculata cells where it catalyzes the conversion of 11-deoxycortisol to cor- tisol; P450c11AS (aldosterone synthase) is found in zona glomerul- osa cells where it catalyzes the three distinct reactions needed to convert deoxycorticosterone to aldosterone ( White et al., 1994; Fardella and Miller, 1996; Miller and Auchus, 2011 ). These two proteins share 93% amino acid sequence identity and are encoded by duplicated genes termed CYP11B1 (producing P450c11b) and CYP11B2
(producing P450c11AS). Like P450scc, both proteins have typical mitochondrial targeting sequence and are associated with the IMM. These enzymes must compete with P450scc for reducing equivalents provided via ferredoxin reductase and ferredoxin. Mutations in CYP11B1 cause 11b-hydroxylase deficiency, a rare form of virilizing congenital adrenal hyperplasia in which the over- production of deoxycorticosterone may lead to mineralocorticoid hypertension. Mutations in CYP11B2 cause aldosterone synthase deficiency. An unusual recombination between the CYP11B1 and CYP11B2
genes can place the CYP11B1 promoter upstream from the CYP11B2 gene, thus producing aldosterone synthase in re- sponse to
causing glucocorticoid suppressible hyperaldosteronism. 11. Mitochondrial P450 enzymes in vitamin D synthesis Vitamin D and its metabolites are not steroids in the strict chemical sense, as the B ring of cholesterol is opened ( Fig. 4
). Nev- ertheless, these sterols are derived from cholesterol, assume shapes that are very similar to steroids, and bind to a similar, zinc-finger receptor that regulates gene transcription ( Feldman et al., 2013 ). The final step in the biosynthesis of cholesterol is con- version of 7-dehydrocholesterol to cholesterol. In human skin, ultraviolet radiation at 270–290 nm directly cleaves the 9–10 car- bon–carbon bond of the cholesterol B ring, converting 7-dehydro- cholesterol to cholecalciferol (vitamin D 3 ) ( Norman, 1998 ). Plants produce ergocalciferol (vitamin D 2 ), which has essentially the same properties as cholecalciferol. Both calciferols are biologically inac- tive pro-hormones that are then activated, and subsequently inac- tivated, by mitochondrial P450 enzymes. The initial step in the activation of vitamin D is its hepatic 25- hydroxylation to 25(OH)D, which may be catalyzed by several en- zymes. The principal 25-hydroxylase is CYP2R1 ( Cheng et al., 2003
), and deficiency of this enzyme causes the very rare 25- hydroxylase deficiency syndrome ( Cheng et al., 2004; Dong and Miller, 2004 ). A hepatic mitochondrial P450 (variously termed P450c25 and P450c27) encoded by the CYP27A1 gene also has vita- min D 25-hydroxylase activity, but its mutation causes cerebroten- dinous xanthomatosis without a disorder in calcium metabolism ( Cali et al., 1991; Leitersdorf et al., 1993 ), hence this mitochondrial enzyme is of marginal importance to vitamin D metabolism. The active, hormonal form of vitamin D, 1,25(OH) 2 D, is pro- duced by the 1 a -hydroxylation of 25(OH)D by the mitochondrial 1 a -hydroxylase, P450c1 a , encoded by the CYP27B1 gene ( Fu et al., 1997a,b ). 1,25(OH) 2 D in the circulation derives primarily W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 69
from the kidney, but 1 a -hydroxylase activity is also found in kerat- inocytes, macrophages, osteoblasts and placenta. 1 a -hydroxylation is the rate-limiting step in the activation of vitamin D, and renal enzyme activity is tightly regulated by parathyroid hormone (PTH), calcium, phosphorus, and 1,25(OH) 2 D itself. The first human clone was obtained from human keratinocytes, but multiple stud- ies, including finding mutations in a patient affected with renal 1 a -
in kidney ( Fu et al., 1997a ). Mutations in this gene cause the disor- der variously termed ‘vitamin D-dependent rickets, Type 1’, ‘pseu- do vitamin D-deficient rickets’ and ‘vitamin D 1 a -hydroxylase deficiency’ ( Wang et al., 1998; Kim et al., 2007 ). The single CYP27B1 gene for 1 a -hydroxylase is only 5 kb long and has an intron/exon organization that is very similar to that of other mitochondrial P450 enzymes, especially the mitochondrial cholesterol side-chain cleavage enzyme, P450scc ( Fu et al., 1997b ). Thus, even though the mitochondrial P450 enzymes retain only 30–40% amino acid se- quence identity with each other, they all belong to a single evolu- tionary lineage. More than 100 patients with CYP27B1 mutations have been described ( Edouard et al 2011 ). 1,25(OH)
2 D may be inactivated by the principal hepatic drug- metabolizing enzyme, microsomal CYP3A4, or by its 24-hydroxyl- ation by vitamin D 24-hydroxylase (P450c24), encoded by the CYP24A1 gene (
Feldman et al., 2013 ). This mitochondrial enzyme can catalyze the 24-hydroxylation of 25(OH)D to 24,25(OH) 2 D and of 1,25(OH) 2 D to 1,24,25(OH) 3 D, primarily in the kidney and intestine, thus inactivating vitamin D ( Ohyama et al., 1991; Chen et al., 1993 ). The expression of P450c24 is induced by 1,25(OH) 2 D, providing a direct feedback mechanism to autoregu- late circulating levels of 1,25(OH) 2 D ( Xie et al., 2002 ). The crystal structure of rat P450c24 has been determined, showing a classic Download 2.44 Mb. Do'stlaringiz bilan baham: 
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