RESUMEN
Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.
Asunto(s)
Uroporfirinógeno Descarboxilasa/metabolismo , Adulto , Anciano , Dominio Catalítico , Cristalografía por Rayos X , Femenino , Genotipo , Heterocigoto , Humanos , Cinética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Porfiria Cutánea Tardía/etiología , Porfiria Cutánea Tardía/genética , Estabilidad Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Uroporfirinógeno Descarboxilasa/química , Uroporfirinógeno Descarboxilasa/genéticaRESUMEN
Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.
Asunto(s)
Modelos Moleculares , Uroporfirinógeno III Sintetasa/química , Sitios de Unión/fisiología , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Porfiria Eritropoyética/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Uroporfirinógeno III Sintetasa/genética , Uroporfirinógeno III Sintetasa/fisiologíaRESUMEN
Oral contraceptives and postmenopausal estrogen replacement therapy are recognized as risk factors for the development of porphyria cutanea tarda (PCT) in women. The recommended clinical practice is to withhold estrogen therapy in women who have had phlebotomy therapy for PCT and are clinically and biochemically normal. We tested the safety and efficacy of transdermal estrogen replacement therapy in 7 women previously treated for PCT and compared them with 19 non-porphyric control subjects treated with transdermal or oral estrogens. Gonadotrophic hormone levels, estrogen levels, liver function studies, body iron stores, urine porphyrin excretion, and cytochrome P4501A2 (CYP1A2) activity were monitored for 1 year. Four of the women previously treated for PCT completed the study. None had evidence of a porphyric relapse. CYP1A2 activity, measured by three different methods, did not differ between study subjects receiving estrogens, patients with active PCT, and non-porphyric control subjects, nor did CYP1A2 activity change during the study period. Gonadotrophic hormone levels fell and estrogen levels rose in all women receiving estrogens. The administration of estrogens by the transdermal route appeared to be safe in the small number of subjects we studied and should be considered for women previously treated for PCT.
Asunto(s)
Terapia de Reemplazo de Estrógeno/efectos adversos , Porfiria Cutánea Tardía/etiología , Porfiria Cutánea Tardía/terapia , Administración Cutánea , Adulto , Cafeína/sangre , Estudios de Casos y Controles , Citocromo P-450 CYP1A2/metabolismo , Femenino , Humanos , Hígado/enzimología , Menopausia , Persona de Mediana Edad , Flebotomía , Porfiria Cutánea Tardía/metabolismo , Recurrencia , Factores de Riesgo , SeguridadRESUMEN
In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Daño del ADN , Metalotioneína/metabolismo , Óxido Nítrico/fisiología , Óxido Nítrico/toxicidad , Células 3T3 , Aminoácido Oxidorreductasas/biosíntesis , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Clonales , Espectroscopía de Resonancia por Spin del Electrón , Expresión Génica , Humanos , Metalotioneína/biosíntesis , Ratones , Óxido Nítrico Sintasa , Compuestos Nitrosos/farmacología , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
We report here that, like nonheme iron, protein-bound intracellular heme iron is also a target for destruction by endogenously produced nitric oxide (NO). In isolated rat hepatocytes NO synthesis results in substantial (approximately 60%) and comparable loss of catalase and cytochrome P450 as well as total microsomal heme, and decreased heme synthetic (delta-aminolevulinate synthetase and ferrochelatase) and increased degradative (heme oxygenase) enzymatic activities. The effect is reversible, and intact cytochrome P450 apoproteins are still present, as judged by heme reconstitution of isolated microsomes. The effects on delta-aminolevulinate synthetase and heme oxygenase are likely to be secondary to heme liberation, while the effects on ferrochelatase appear to be a direct effect of NO, perhaps destruction of its nonheme iron-sulfur center.
Asunto(s)
Catalasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Citocinas/farmacología , Hemo/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/enzimología , Óxido Nítrico/biosíntesis , Vasodilatadores/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Células Cultivadas , Hierro/metabolismo , Hígado/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Óxido Nítrico/farmacología , Óxido Nítrico/fisiología , Penicilamina/análogos & derivados , Penicilamina/farmacología , ARN Mensajero/metabolismo , S-Nitroso-N-Acetilpenicilamina , omega-N-MetilargininaRESUMEN
Figure 2 depicts a working hypothesis for these results. Activation of .NO synthesis results in nitrogen oxide-induced loss of protein-bound heme from CYP proteins, which remain relatively intact. This heme liberation results in a decrease in heme synthesis (decreased ALAS) and an increase in heme degradation (increased HO). In addition, .NO synthesis results in direct inhibition of ferrochelatase, which further contributes to inhibition of heme synthesis. There also appears to be a mechanism to repair or resynthesize CYP after .NO synthesis is inhibited. Finally, a result of this effect may be protection against cellular injury, since increased HO is an important response against cellular injury from a variety of insults.
Asunto(s)
Hemo/fisiología , Óxido Nítrico/fisiología , Animales , Catalasa/biosíntesis , Citocromos/biosíntesis , Hemo/metabolismo , Humanos , Óxido Nítrico/biosíntesis , Óxido Nítrico/farmacologíaRESUMEN
Isolated rat hepatocytes were examined by EPR spectroscopy after exposure to inflammatory stimuli (interferon-gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and lipopolysaccharide [LPS]) in vitro, after in vivo immune activation by Corynebacterium parvum, and after exposure to .N = O and to nitroprusside (nitroferricyanide), an NO-donating nitrovasodilator. Hepatocytes exposed to IFN-gamma, TNF-alpha, IL-1 beta, and LPS demonstrated the appearance of a g = 2.04 axial EPR signal indicative of the formation of nonheme iron-nitrosyl complexes. Concurrent incubation with L-NG-monomethylarginine (L-NMMA), a competitive inhibitor of .N = O synthase, prevented the appearance of the signal. The g = 2.04 signal was localized in the cytosolic fraction of hepatocyte extracts. Hepatocytes freshly isolated from C. parvum-treated rats exhibited a modest g = 2.04 signal, which was increased by a factor of approximately 2.5-fold upon subsequent 24-h culture in media without additional stimuli. This increase was prevented by L-NMMA in the culture medium and also by the presence of rat erythrocytes added to the culture. In the presence of erythrocytes, virtually all of the .N = O produced was oxidized by reaction with intracellular hemoglobin within the erythrocyte, as judged by the relative amounts of nitrite and nitrate detected. These results suggest that in this model system .N = O is sufficiently stable and diffusible to escape from the hepatocyte and diffuse into the erythrocyte without first reacting with oxygen or with intracellular iron at the site of its formation within the hepatocyte. Treatment of hepatocytes with exogenous .N = O or nitroprusside generated an identical g = 2.04 signal of much greater intensity than with cytokines plus LPS. Treatment with nitroprusside also caused the appearance of a signal from pentacyanonitrosylferrate ion, verifying the previously reported metabolism of this nitrovasodilator by reduction and liberation of cyanide ion and .N = O. These results indicate significant differences in intracellular nonheme iron nitrosylation in hepatocytes compared to cytotoxic activated macrophages, which may correlate with the differences in physiological function of .N = O in these two systems.
Asunto(s)
Hierro/metabolismo , Hígado/metabolismo , Óxido Nítrico/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Interferón gamma/farmacología , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Masculino , Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Propionibacterium acnes/inmunología , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/farmacología , omega-N-MetilargininaRESUMEN
BACKGROUND: Our previous observation that nitric oxide (NO) is synthesized during antigen-specific immune reactions in vitro led us to investigate whether NO is produced during the in vivo immune response to a vascularized organ allograft. METHODS: Orthotopic small-bowel transplantation in the rat was performed by standard microsurgical techniques in the LBNF1 to Lewis (rejection alone), Lewis to LBNF1 (graft-versus-host disease [GVHD] alone), and a syngeneic strain combination with and without immunosuppressive therapy with FK 506. The recipient serum NO2-/NO3- levels (stable end products of NO metabolism) were measured and erythrocytes were evaluated for the presence of nitrosylferrohemoglobin (specific for NO bound to hemoglobin). RESULTS: Animals that acutely rejected small-bowel allografts or suffered from acute GVHD showed significantly elevated serum NO2-/NO3- levels on days 6 and 9, and nitrosylferrohemoglobin electron paramagnetic resonance signals of different intensity were detected on days 3, 6, and 9. FK 506-treated allograft recipients and recipients of syngeneic grafts showed normal serum NO2-/NO3- levels and lacked nitrosylferrohemoglobin signals at all time points. CONCLUSIONS: This study indicates that NO is produced early during the course of small-bowel allograft rejection and GVHD and might therefore serve as a simple marker to detect such immune reactions.
Asunto(s)
Rechazo de Injerto , Enfermedad Injerto contra Huésped/metabolismo , Intestino Delgado/trasplante , Óxido Nítrico/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/patología , Intestino Delgado/patología , Masculino , Óxido Nítrico/sangre , Óxido Nitroso/sangre , Complicaciones Posoperatorias , Ratas , Ratas Endogámicas Lew , Ratas EndogámicasRESUMEN
The paramagnetic molecule nitric oxide (NO), produced from L-arginine by a specific enzyme (NO synthase), has been shown to be involved in a surprising variety of mammalian cellular responses, including the regulation of T cell immunity to alloantigens in vitro. In cytotoxic activated macrophages, NO production results in a characteristic pattern of alteration of iron-containing enzyme function that is mimicked by exposure to NO. Electron paramagnetic resonance (EPR) studies have shown the formation of iron-nitrosyl species during macrophage activation and also during sepsis, indicating that alteration of iron-containing protein function may be the result of the well-documented tendency of NO to bind to metal ions. We have recently shown that the NO synthesis induced during alloantigenic activation of rat splenocytes inhibits lymphocyte proliferation and cytotoxic T-lymphocyte generation. This report demonstrates that iron-nitrosyl EPR signals similar to those observed in macrophages and during sepsis are present in the blood and in the grafted tissue of rats during the rejection of allogeneic (but not syngeneic) heart grafts. These signals are found in the blood and at the site of allograft rejection, but are not found in other tissues (such as spleen and lung), and are obliterated by administration of the immunosuppressant FK506. These results directly demonstrate the formation of iron-nitrosyl complexes during vascularized allograft rejection and suggest that consequent destruction of iron-containing protein function plays an important role in the rejection response.