RESUMEN
The human adrenal cortex secretes aldosterone and cortisol as major corticosteroids. For their production, CYP11B2 and CYP11B1 catalyze the last steps in the syntheses of aldosterone and cortisol, respectively. In our previous study, CYP11B2 was the first successfully purified from rat adrenals and human clinical samples and then was proved to be aldosterone synthase. We demonstrated the immunohistochemistry for CYP11B2 of both rats and humans and applied it clinically to visualize the functional histology of aldosterone-producing adenoma (APA) causing primary aldosteronism (PA). We discovered aldosterone-producing cell clusters (APCCs) and possible APCC-to-APA transitional lesions (pAATLs) and further visualized aldosterone-producing lesions for rare forms of PA including familial hyperaldosteronism type 3 and novel non-familial juvenile PA. Here we review the history of our research on aldosterone-producing lesions.
Asunto(s)
Neoplasias de la Corteza Suprarrenal , Hiperaldosteronismo , Humanos , Animales , Ratas , Aldosterona/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Hidrocortisona , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , MutaciónRESUMEN
Our group previously purified human and rat aldosterone synthase (CYP11B2 and Cyp11b2, respectively) from their adrenals and verified that it is distinct from steroid 11ß-hydroxylase (CYP11B1 or Cyp11b1), the cortisol- or corticosterone-synthesizing enzyme. We now describe their distributions immunohistochemically with specific antibodies. In rats, there is layered functional zonation with the Cyp11b2-positive zona glomerulosa (ZG), Cyp11b1-positive zona fasciculata (ZF), and Cyp11b2/Cyp11b1-negative undifferentiated zone between the ZG and ZF. In human infants and children (<12 years old), the functional zonation is similar to that in rats. In adults, the adrenal cortex remodels and subcapsular aldosterone-producing cell clusters (APCCs) replace the continuous ZG layer. We recently reported possible APCC-to-APA transitional lesions (pAATLs) in 2 cases of unilateral multiple adrenocortical micro-nodules. In this review, we present 4 additional cases of primary aldosteronism, from which the extracted adrenals contain pAATLs, with results of next generation sequencing for these lesions. Immunohistochemistry for CYP11B2 and CYP11B1 has become an important tool for the diagnosis of and research on adrenocortical pathological conditions and suggests that APCCs may be the origin of aldosterone-producing adenoma.
Asunto(s)
Citocromo P-450 CYP11B2/metabolismo , Hiperaldosteronismo/enzimología , Hiperaldosteronismo/patología , Inmunohistoquímica/métodos , Aldosterona/biosíntesis , Animales , Biocatálisis , Citocromo P-450 CYP11B2/genética , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
BACKGROUND: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes. METHODS: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids. RESULTS: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients. CONCLUSIONS: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Estradiol/farmacología , Progesterona/farmacología , Aldosterona/metabolismo , Citocromo P-450 CYP11B2/antagonistas & inhibidores , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Células HEK293 , Humanos , Hiperaldosteronismo/genética , Cinética , Proteínas Mutantes Quiméricas/metabolismoRESUMEN
BACKGROUND: In primary aldosteronism (PA) the main source of aldosterone hypersecretion is an aldosterone-producing adenoma (APA) or a bilateral hyperplasia. Histopathology of the adrenal gland from patients with PA has been difficult, as there are no morphological criteria to ascertain which are the cells that produce aldosterone. We therefore applied new specific antibodies to explore which cells in the adrenal contain the enzymes for aldosterone and cortisol production, respectively. METHODS: Adrenals from 24 patients with PA were studied. After routine preparation, consecutive sections were stained with antibodies for CYP11B1 (cortisol) and CYP11B2 (aldosterone) enzymes. RESULTS: APA had a strong immunoreactivity for CYB11B2. In adrenals from seven patients, we found no APA, but several nodules with strong CYB11B2 immunoreactivity, indicating aldosterone-producing nodular hyperplasia. CONCLUSIONS: Immunohistochemistry of adrenal steroidogenic enzymes provides novel diagnostic information. This may become an important part of routine histopathology, and contribute to improved clinical management in PA.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Glándulas Suprarrenales/enzimología , Adenoma Corticosuprarrenal/enzimología , Citocromo P-450 CYP11B2/análisis , Hiperaldosteronismo/enzimología , Inmunohistoquímica , Esteroide 11-beta-Hidroxilasa/análisis , Neoplasias de la Corteza Suprarrenal/complicaciones , Neoplasias de la Corteza Suprarrenal/patología , Glándulas Suprarrenales/patología , Adenoma Corticosuprarrenal/complicaciones , Adenoma Corticosuprarrenal/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Femenino , Humanos , Hiperaldosteronismo/etiología , Hiperaldosteronismo/patología , Hiperplasia , Masculino , Persona de Mediana Edad , Valor Predictivo de las PruebasRESUMEN
The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.
Asunto(s)
Córnea/efectos de los fármacos , Síndromes de Ojo Seco/tratamiento farmacológico , Células Epiteliales/efectos de los fármacos , Epitelio Corneal/efectos de los fármacos , Lactoferrina/farmacología , Selenio/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Células Cultivadas , Córnea/metabolismo , Córnea/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/antagonistas & inhibidores , Desoxiguanosina/biosíntesis , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Interleucina-6/agonistas , Interleucina-6/genética , Interleucina-6/metabolismo , Aparato Lagrimal/cirugía , Lactoferrina/química , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Soluciones Oftálmicas , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Selenio/químicaRESUMEN
CONTEXT: Aldosterone synthase (CYP11B2) and steroid 11 beta-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiation of human adrenocortical cells. Little is known, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. OBJECTIVE: The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. RESULTS: Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster, and a CYP11B1-expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushing's syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. CONCLUSION: Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas.
Asunto(s)
Corteza Suprarrenal/patología , Corteza Suprarrenal/fisiología , Corteza Suprarrenal/enzimología , Aldosterona/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Carcinoma de Células Renales/enzimología , Corticosterona/metabolismo , Síndrome de Cushing/enzimología , Citocromo P-450 CYP11B2/deficiencia , Citocromo P-450 CYP11B2/metabolismo , Humanos , Neoplasias Renales/enzimología , Mamíferos , Fragmentos de Péptidos/química , Conejos , Valores de Referencia , Roedores , Esteroide 11-beta-Hidroxilasa/metabolismo , Zona Fascicular/enzimología , Zona Glomerular/enzimologíaRESUMEN
We have found cytochrome P-450(17alpha) in the islets of Langerhans of rat pancreas. Its existence coincided with that of insulin and demarcated those of glucagon and somatostatin, demonstrating the localization in beta-cells. The enzyme has not only 17alpha-hydroxylase activity but also lyase one, which is a prerequisite for androgen biosynthesis. The pancreatic microsomes converted progesterone mainly to androstenedione with a minor production of 17alpha-hydroxyprogesterone. Due to a low activity of the built-in lyase, cytochrome P-450(17alpha) requires a sufficient electron-transfer from P-450 reductase or presence of an activator to promote the C-C bond cleavage. In beta-cells, P-450 reductase was abundant and could efficiently transfer electrons to P-450(17alpha). Actually, inhibition with anti-P-450 reductase or limitation of NADPH preferentially reduced the lyase activity. Androstenedione was accumulated when its further metabolism was suppressed. We also found localization of cytochrome P-450scc and 3beta-hydroxysteroid dehydrogenase in beta-cells. These results indicate that the immediate substrate for androgen formation, progesterone, is intracellularly produced and is converted mainly to androstenedione with support by an efficient electron supply from P-450 reductase. The product was supposed to be further metabolized to the reduced derivatives such as testosterone, 5alpha-androstanedione, and dihydrotestosterone, which would act as local steroids in the islets of Langerhans.
Asunto(s)
Células Secretoras de Insulina/enzimología , Páncreas/citología , Páncreas/enzimología , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroides/biosíntesis , Animales , Inmunohistoquímica , Masculino , Microsomas/enzimología , Ratas , Ratas Sprague-Dawley , Esteroides/análisisRESUMEN
ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.
Asunto(s)
Glándulas Suprarrenales/crecimiento & desarrollo , Gluconeogénesis/fisiología , Receptor de Melanocortina Tipo 2/fisiología , Esteroides/biosíntesis , Animales , Animales Recién Nacidos , Ratones , Ratones Noqueados , Receptor de Melanocortina Tipo 2/genéticaRESUMEN
Protein turnover, which occurs at various rates, is critical for the homeostasis of cellular protein levels. However, the proteolysis systems that determine the turnover rate of mitochondrial proteins are largely unknown. Delta-aminolevulinic acid synthase (ALAS) 1, a rate-limiting enzyme in the haeme biosynthesis, is one of the mitochondrial proteins that have a very short lifetime. In this study, to reveal the regulatory mechanisms for ALAS1 degradation, we examined the turnover rates of ALAS1 in rat liver under several conditions. In primary rat hepatocytes, the degradation of ALAS1 was stimulated by haeme, and suppressed by inhibition of haeme biosynthesis. Furthermore, the haeme-stimulated degradation of ALAS1 was observed in the isolated mitochondria. These results suggested that, in mitochondria, there exists an ALAS1 degradation system that is regulated by cellular haeme level and plays a crucial role in the regulation of haeme biosynthesis.
Asunto(s)
5-Aminolevulinato Sintetasa/metabolismo , Hemo/fisiología , 5-Aminolevulinato Sintetasa/biosíntesis , 5-Aminolevulinato Sintetasa/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Hemo/antagonistas & inhibidores , Hemo/biosíntesis , Masculino , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Metaloendopeptidasas/química , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Colorantes Fluorescentes/farmacología , Glicina/química , Proteínas Fluorescentes Verdes/metabolismo , Lisina/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina/química , Peptidasa de Procesamiento MitocondrialRESUMEN
The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the fetal expression of testicular cytochrome P450 17 (CYP17), one of the enzymes necessary for sex steroid synthesis, was studied in Wistar rats. Fetal testicular CYP17 exhibited reduced mRNA and protein levels following exposure of the dams at gestational day 15 to 1 microg/kg TCDD. In support of this, CYP17 activity catalyzed by fetal testis homogenate was also reduced by maternal exposure to TCDD. The reduction in CYP17 expression seemed to be specific for fetal stages, because 7 day-old pups born from TCDD-treated dams did not exhibit any reduction in CYP17. In sharp contrast to the in vivo observations, TCDD failed to reduce CYP17 expression in cultured fetal testis, although CYP17 could be induced by activating cAMP-dependent signaling. To assess the role of pituitary luteinizing hormone (LH) on TCDD-induced reduction in fetal testicular CYP17, a further investigation was performed to examine whether the direct injection of LH into fetuses restores the altered CYP17 expression. The results showed that in utero injection of equine chorionic gonadotropin, an LH-mimicking hormone, completely abolishes the TCDD-produced reduction in fetal CYP17. However, neither the alpha- nor beta-subunits of LH in cultured fetal pituitary was reduced by TCDD. These results suggest that 1) maternal exposure to TCDD impairs the expression of testicular CYP17 in a fetal stage-specific manner; 2) this effect is due, at least partially, to a TCDD-produced reduction in circulating LH; and 3) TCDD exerts such an effect by affecting the upstream mechanism regulating the pituitary synthesis of LH.
Asunto(s)
Contaminantes Ambientales/toxicidad , Hormona Luteinizante/biosíntesis , Hipófisis/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Testículo/enzimología , Animales , AMP Cíclico/biosíntesis , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Gonadotropinas Equinas/farmacología , Inyecciones , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Exposición Materna , Técnicas de Cultivo de Órganos , Hipófisis/embriología , Hipófisis/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Esteroide 17-alfa-Hidroxilasa/biosíntesis , Esteroide 17-alfa-Hidroxilasa/genética , Testículo/embriologíaRESUMEN
The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the alpha and beta subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with beta-MPP but barely contained any of the glycine-rich loop region characteristic of alpha-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast beta-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Rickettsia prowazekii/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cinética , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Filogenia , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rickettsia prowazekii/genética , Alineación de Secuencia , Peptidasa de Procesamiento MitocondrialRESUMEN
It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.
Asunto(s)
Ácido Ascórbico/química , Oxigenasas de Función Mixta/fisiología , Esteroides/biosíntesis , Zona Glomerular/enzimología , Animales , Ácido Ascórbico/fisiología , Citocromos b/fisiología , Masculino , Membranas Mitocondriales/enzimología , Oxigenasas de Función Mixta/metabolismo , Ratas , Ratas Endogámicas WKY , Ratas Mutantes , Ratas Sprague-DawleyRESUMEN
The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence.
Asunto(s)
Núcleo Celular/genética , Metaloendopeptidasas/metabolismo , Mitocondrias/enzimología , Poliproteínas/metabolismo , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/enzimología , Metaloendopeptidasas/genética , Mitocondrias/metabolismo , Mutación/genética , Oryza , Poliproteínas/química , Poliproteínas/genética , Pliegue de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Transporte de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Succinato Deshidrogenasa/química , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Peptidasa de Procesamiento MitocondrialRESUMEN
Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017alpha, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017alpha-catalyzed reactions, 17alpha-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17alpha-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17alpha-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells.