RESUMO
Diabetes mellitus is characterized by insulin resistance and ß-cell failure. The latter involves impaired insulin secretion and ß-cell dedifferentiation. Sulfonylurea (SU) is used to improve insulin secretion in diabetes, but it suffers from secondary failure. The relationship between SU secondary failure and ß-cell dedifferentiation has not been examined. Using a model of SU secondary failure, we have previously shown that functional loss of oxidoreductase Cyb5r3 mediates effects of SU failure through interactions with glucokinase. Here we demonstrate that SU failure is associated with partial ß-cell dedifferentiation. Cyb5r3 knockout mice show more pronounced ß-cell dedifferentiation and glucose intolerance after chronic SU administration, high-fat diet feeding, and during aging. A Cyb5r3 activator improves impaired insulin secretion caused by chronic SU treatment, but not ß-cell dedifferentiation. We conclude that chronic SU administration affects progression of ß-cell dedifferentiation and that Cyb5r3 activation reverses secondary failure to SU without restoring ß-cell dedifferentiation.
Assuntos
Citocromo-B(5) Redutase , Diabetes Mellitus Tipo 2 , Resistência à Insulina , Células Secretoras de Insulina , Animais , Camundongos , Desdiferenciação Celular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina/farmacologia , Compostos de Sulfonilureia/farmacologia , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismoRESUMO
Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.
Assuntos
Neoplasias Pulmonares , Proteínas Serina-Treonina Quinases , Animais , Humanos , Camundongos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/genética , Citocromo-B(5) Redutase/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5 ) and cytochrome b5 reductase (b5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5 R domains (CS/b5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34 MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116 QMDWLKLTRT126 ) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes.
Assuntos
Citocromos b , NAD , Animais , Humanos , Citocromo-B(5) Redutase/química , Oxirredutases , Heme/químicaRESUMO
Cytochrome b5 reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme, mimicking some of the beneficial effects of calorie restriction. The aim of our study was to investigate the role of sex on metabolic adaptations elicited by CYB5R3 overexpression, and how key markers related with mitochondrial function are modulated in skeletal muscle, one of the major contributors to resting energy expenditure. Young CYB5R3 transgenic mice did not exhibit the striking adaptations in carbon metabolism previously detected in older animals. CYB5R3 was efficiently overexpressed and targeted to mitochondria in skeletal muscle from transgenic mice regardless sex. Overexpression significantly elevated NADH in both sexes, although differences were not statistically significant for NAD+, and increased the abundance of cytochrome c and the fission protein DRP-1 in females but not in males. Moreover, while mitochondrial biogenesis and function markers (as TFAM, NRF-1 and cleaved SIRT3) were markedly upregulated by CYB5R3 overexpression in females, a downregulation was observed in males. Ultrastructural changes were also highlighted, with an increase in the number of mitochondria per surface unit, and in the size of intermyofibrillar mitochondria in transgenic females compared with their wild-type controls. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial biogenesis and function, and increases mitochondrial abundance in skeletal muscle, producing most of these potentially beneficial actions in females.
Assuntos
Citocromo-B(5) Redutase , Mitocôndrias , Animais , Feminino , Masculino , Camundongos , Proteínas de Transporte/metabolismo , Citocromo-B(5) Redutase/química , Citocromo-B(5) Redutase/metabolismo , Metabolismo Energético/genética , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Fatores SexuaisRESUMO
Background: Cardiopulmonary disorders are the most common cause of central cyanosis, and methemoglobinemia is often overlooked in the differential diagnosis of patients with central cyanosis. In most cases, methemoglobinemia is acquired and hereditary congenital methemoglobinemia is rare. Only a few case reports of congenital methemoglobinemia can be found in PubMed. To date, only four cases of congenital methemoglobinemia diagnosed after the age of 50 years have been reported. Case Presentation: A 79-year-old Japanese woman presented at our hospital with the chief complaints of dyspnea and cyanosis. She exhibited cyanosis of the lips and extremities, and her SpO2 was 80%, with oxygen administration at 5 L/min. Blood gas analysis revealed a PaO2 of 325.4 mmHg and methemoglobin level of 36.9%. The SpO2 and PaO2 values were dissociated, and methemoglobin levels were markedly elevated. Genetic analysis revealed a nonsynonymous variant in the gene encoding nicotinamide adenine dinucleotide cytochrome (NADH) B5 reductase 3 (CYB5R3), and the patient was diagnosed with congenital methemoglobinemia. Conclusions: It is important to consider methemoglobinemia in the differential diagnosis of patients with central cyanosis. At 79 years of age, our patient represents the oldest patient with this diagnosis. This report indicates that it is crucial to consider the possibility of methemoglobinemia regardless of the patient's age.
Assuntos
Metemoglobinemia , Humanos , Feminino , Idoso , Pessoa de Meia-Idade , Metemoglobinemia/diagnóstico , Metemoglobinemia/genética , Metemoglobinemia/congênito , Metemoglobina/análise , Citocromo-B(5) Redutase/genética , Cianose/genéticaRESUMO
Sulfonylureas (SUs) are effective and affordable antidiabetic drugs. However, chronic use leads to secondary failure, limiting their utilization. Here, we identify cytochrome b5 reductase 3 (Cyb5r3) down-regulation as a mechanism of secondary SU failure and successfully reverse it. Chronic exposure to SU lowered Cyb5r3 abundance and reduced islet glucose utilization in mice in vivo and in ex vivo murine islets. Cyb5r3 ß cell-specific knockout mice phenocopied SU failure. Cyb5r3 engaged in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Hence, Gck activators can circumvent Cyb5r3-dependent SU failure. A Cyb5r3 activator rescued secondary SU failure in mice in vivo and restored insulin secretion in ex vivo human islets. We conclude that Cyb5r3 is a key factor in the secondary failure to SU and a potential target for its prevention, which might rehabilitate SU use in diabetes.
Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Camundongos , Humanos , Animais , Compostos de Sulfonilureia/farmacologia , Compostos de Sulfonilureia/uso terapêutico , Glucose , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Citocromo-B(5) RedutaseRESUMO
Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-ß1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-ß1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.
Assuntos
Células Epiteliais Alveolares , Fibrose Pulmonar Idiopática , Humanos , Células Epiteliais Alveolares/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Transdução de Sinais , Citocromo-B(5) Redutase/metabolismoRESUMO
Background and Objective: Mutations in the CYB5R3 gene cause reduced NADH-dependent cytochrome b5 reductase enzyme function and consequently lead to recessive congenital methemoglobinemia (RCM). RCM exists as RCM type I (RCM1) and RCM type II (RCM2). RCM1 leads to higher methemoglobin levels causing only cyanosis, while in RCM2, neurological complications are also present along with cyanosis. Materials and Methods: In the current study, a consanguineous Pakistani family with three individuals showing clinical manifestations of cyanosis, chest pain radiating to the left arm, dyspnea, orthopnea, and hemoptysis was studied. Following clinical assessment, a search for the causative gene was performed using whole exome sequencing (WES) and Sanger sequencing. Various variant effect prediction tools and ACMG criteria were applied to interpret the pathogenicity of the prioritized variants. Molecular dynamic simulation studies of wild and mutant systems were performed to determine the stability of the mutant CYB5R3 protein. Results: Data analysis of WES revealed a novel homozygous missense variant NM_001171660.2: c.670A > T: NP_001165131.1: p.(Ile224Phe) in exon 8 of the CYB5R3 gene located on chromosome 22q13.2. Sanger sequencing validated the segregation of the identified variant with the disease phenotype within the family. Bioinformatics prediction tools and ACMG guidelines predicted the identified variant p.(Ile224Phe) as disease-causing and likely pathogenic, respectively. Molecular dynamics study revealed that the variant p.(Ile224Phe) in the CYB5R3 resides in the NADH domain of the protein, the aberrant function of which is detrimental. Conclusions: The present study expanded the variant spectrum of the CYB5R3 gene. This will facilitate genetic counselling of the same and other similar families carrying mutations in the CYB5R3 gene.
Assuntos
Metemoglobinemia , Humanos , Metemoglobinemia/congênito , Metemoglobinemia/genética , Simulação de Dinâmica Molecular , NAD/genética , NAD/metabolismo , Mutação , Cianose , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismoRESUMO
OBJECTIVE: To determine whether dogs with cytochrome b5 reductase (CYB5R) deficiency have a constitutive proinflammatory phenotype, characterize hematologic and serum chemistry results, and describe changes in methemoglobin (MetHb) levels and serum C-reactive protein (CRP) concentrations after long-term per os (PO) methylene blue (MB) therapy. ANIMALS: 21 client-owned dogs (CYB5R deficient, n = 10; healthy controls, 11). PROCEDURES: In this prospective, case-control study, methemoglobin levels were measured using a blood gas analyzer with co-oximetry. Plasma tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations were measured using a canine-specific multiplex bead-based assay. Serum CRP concentrations were measured with a canine-specific commercial ELISA kit. Serum CRP concentration and MetHb levels were measured in 6 dogs with CYB5R deficiency after ≥ 60 days of PO MB therapy. RESULTS: As expected, MetHb levels were higher in dogs with CYB5R deficiency compared to controls (P < .001). Plasma TNF-α, IL-6, IL-10, and serum CRP concentrations were no different between CYB5R-deficient and control dogs. Dogs with CYB5R deficiency had lower absolute lymphocyte (P = .005) and eosinophil counts (P = .04) and higher alanine transaminase (P = .04) and alkaline phosphatase activity (P = .02) than controls, but these changes were not clinically relevant. Methemoglobin levels decreased after PO MB therapy (P = .03). CLINICAL RELEVANCE: These results suggest that otherwise healthy dogs with CYB5R deficiency do not have a constitutive proinflammatory phenotype and clinically relevant abnormalities in hematologic and serum chemistry panels are not expected. Dogs with decreased quality of life attributed to methemoglobinemia from CYB5R deficiency might benefit from PO MB therapy.
Assuntos
Doenças do Cão , Metemoglobinemia , Cães , Animais , Metemoglobinemia/veterinária , Metemoglobinemia/tratamento farmacológico , Metemoglobinemia/genética , Azul de Metileno/uso terapêutico , Metemoglobina/genética , Metemoglobina/metabolismo , Metemoglobina/uso terapêutico , Interleucina-10/genética , Interleucina-10/uso terapêutico , Citocromos b5/genética , Interleucina-6/genética , Interleucina-6/uso terapêutico , Fator de Necrose Tumoral alfa/genética , Estudos de Casos e Controles , Estudos Prospectivos , Qualidade de Vida , Citocromo-B(5) Redutase/genética , Fenótipo , Doenças do Cão/tratamento farmacológico , Doenças do Cão/genéticaRESUMO
Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.
Assuntos
Autofagia , Citocromo-B(5) Redutase , Retículo Endoplasmático , Ubiquitinas , Animais , Camundongos , Autofagia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Citocromo-B(5) Redutase/química , Citocromo-B(5) Redutase/metabolismo , Retículo Endoplasmático/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitinação/fisiologia , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMO
The cytochrome-b5 reductase (CYB5R) family of flavoproteins is known to regulate reduction-oxidation (redox) balance in cells. The five enzyme members are highly compartmentalized at the subcellular level and function as "redox switches" enabling the reduction of several substrates, such as heme and coenzyme Q. Critical insight into the physiological and pathophysiological significance of CYB5R enzymes has been gleaned from several human genetic variants that cause congenital disease and a broad spectrum of chronic human diseases. Among the CYB5R genetic variants, CYB5R3 is well-characterized and deficiency in expression and activity is associated with type II methemoglobinemia, cancer, neurodegenerative disorders, diabetes, and cardiovascular disease. Importantly, pharmacological and genetic-based strategies are underway to target CYB5R3 to circumvent disease onset and mitigate severity. Despite our knowledge of CYB5R3 in human health and disease, the other reductases in the CYB5R family have been understudied, providing an opportunity to unravel critical function(s) for these enzymes in physiology and disease. In this review, we aim to provide the broad scientific community an up-to-date overview of the molecular, cellular, physiological, and pathophysiological roles of CYB5R proteins.
Assuntos
Citocromo-B(5) Redutase , Metemoglobinemia , Humanos , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Metemoglobinemia/congênito , Metemoglobinemia/genética , Oxirredução , Homeostase , Redutases do Citocromo/metabolismoRESUMO
Lignin is a complex heterogenous polymer derived from oxidative radical polymerization of three monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignin monomeric precursors structurally differ in their methoxy groups of the benzene rings. In phenylpropanoid-monolignol biosynthetic pathway, the endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, cinnamate 4-hydroxylase, coumaroyl ester 3'-hydroxylase and ferulate 5-hydroxylase, establish the key structural characteristics of monolignols. The catalysis of cytochrome P450 monooxygenase requires reducing power, which is supplied by the ER electron transfer chains, composed of cytochrome P450 oxidoreductase (CPR), cytochrome b5 reductase (CBR) and/or cytochrome b5 protein (CB5), from cofactor NADPH or NADH. While NADPH-dependent CPR serves as the typical electron donor for most P450 enzymes, in some cases, the CBR-CB5 or CPR-CB5 electron transfer system also transfers electrons to the terminal P450 enzymes. There are tremendous studies focusing on the discovery and characterization of cytochrome P450 monooxygenases. However, very limited attention has been paid to the versatility and the roles of electron transfer components in the P450 catalytic system. Due to the membrane-residence property of both P450 enzymes and electron transfer components, it is challenging to establish an effective experimental system to evaluate the functional association of P450s with their redox partners. This chapter describes a yeast cell biocatalytic system and the related experimental procedures for comparatively assessing the functional relationship of monolignol biosynthetic P450 enzymes and different redox partners in their catalysis.
Assuntos
Citocromo-B(5) Redutase , Lignina , Citocromo-B(5) Redutase/metabolismo , Lignina/metabolismo , NADP , Transcinamato 4-Mono-Oxigenase/metabolismo , Citocromos b/metabolismo , Benzeno , NAD/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução , ÉsteresRESUMO
Diabetic retinopathy (DR) is a progressive vascular complication of diabetes mellitus (DM) and is related to retinal vascular abnormalities. NADH-Cytochrome B5 Reductase 2 (CBR2) has been implicated in angiogenesis, but the effect of CBR2 on angiogenesis and endothelial cell biological behavior in DR remains unclear. Here, we aimed to explore the effect of CBR2 on retinal vascular dysfunction under diabetic conditions. The histological analyses were performed to explore the effect of CBR2 on pathological change in streptozotocin (STZ)-induced diabetic rat retinas. The effect of CBR2 on endothelial cell function was explored by CCK-8, scratch wound, transwell, tube formation, and immunofluorescence assays in high glucose (HG)-stimulated human retinal microvascular endothelial cells (HRMECs). CBR2 expression was significantly downregulated in DM rat retinas and HG-stimulated HRMECs. Intravitreal injection of CBR2-expressing lentivirus under diabetic conditions reduced retinal angiogenesis, acellular capillary formation, and pericyte loss, along with decreased expression of hypoxia-inducible factor-1α (HIF-1α), cluster of differentiation 31 (CD31), and vascular endothelial growth factor A (VEGFA) in vivo. Moreover, CBR2 overexpression inhibited cell growth and tube formation and led to decreased expression of HIF-1α and VEGFA in HG-induced HRMECs. Interestingly, the repressive effects of CBR2 on cell proliferation, migration, and tube formation under HG conditions were strongly reversed when VEGFA was overexpressed. Overall, the key findings of our study suggested that CBR2 might alleviate retinal vascular dysfunction and abnormal endothelial proliferation during the process of DR by regulating VEGFA, providing a piece of potent evidence for DR therapy.
Assuntos
Citocromo-B(5) Redutase , Diabetes Mellitus , Retinopatia Diabética , Animais , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Diabetes Mellitus/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Humanos , Neovascularização Patológica/metabolismo , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Five molybdenum-dependent enzymes are known in eukaryotes. While four of them are under investigation since decades, the most recently discovered, (mitochondrial) amidoxime reducing component ((m)ARC), has only been characterized in mammals and the green algae Chlamydomonas reinhardtii. While mammalian mARCs have been shown to be involved in various signalling pathways, Chlamydomonas ARC was shown to be a nitric oxide (NO)-forming nitrite reductase. Similar to mammals, higher plants possess two ARC proteins. To test whether plant ARCs have a similar function in NO production to the function they have in C. reinhardtii, we analysed the enzymes from the model plant Arabidopsis thaliana. Both ARC1 and ARC2 from Arabidopsis could reduce N-hydroxylated compounds, while nitrite reduction to form NO could only be demonstrated for ARC2. Searching for physiological electron donors, we found that both ARC enzymes accept electrons from NADH via cytochrome b5 reductase and cytochrome b5 , but only ARC2 is able to accept electrons from nitrate reductase at all. Furthermore, arc-deficient mutant plants were similar to wildtype plants regarding growth and also nitrite-dependent NO-formation. Altogether, our results did not confirm the hypothesis that either ARC1 or ARC2 from Arabidopsis are involved in physiologically relevant nitrite-dependent NO-formation. In contrast, our data suggest that ARC1 and ARC2 have distinct, yet unknown physiological roles in higher plants.
Assuntos
Arabidopsis , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Citocromo-B(5) Redutase/metabolismo , Citocromos b , Mamíferos/metabolismo , Molibdênio/metabolismo , NAD , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Óxido Nítrico/metabolismo , Nitrito Redutases/metabolismo , Nitritos/metabolismo , OximasRESUMO
The endoplasmic reticulum (ER) membrane of plant cells contains several enzymes responsible for the biosynthesis of a diverse range of molecules essential for plant growth and holds potential for industrial applications. Many of these enzymes are dependent on electron transfer proteins to sustain their catalytic cycles. In plants, two crucial ER-bound electron transfer proteins are cytochrome b5 and cytochrome b5 reductase, which catalyze the stepwise transfer of electrons from NADH to redox enzymes such as fatty acid desaturases, cytochrome P450s, and plant aldehyde decarbonylase. Despite the high significance of plant cytochrome b5 and cytochrome b5 reductase, they have eluded detailed characterization to date. Here, we overexpressed the full-length membrane-bound cytochrome b5 isoform B from the model plant Arabidopsis thaliana in Escherichia coli, purified the protein employing detergents as well as styrene-maleic acid (SMA) copolymers, and biochemically characterized the protein. The SMA-encapsulated cytochrome b5 exhibits a discoidal shape and the characteristic features of the active heme-bound state. We also overexpressed and purified the soluble domain of cytochrome b5 reductase from A. thaliana, establishing its activity, stability, and kinetic parameters. Further, we demonstrated that the plant cytochrome b5, purified in detergents and styrene maleic acid lipid particles (SMALPs), readily accepts electrons from the cognate plant cytochrome b5 reductase and distant electron mediators such as plant NADPH-cytochrome P450 oxidoreductase and cyanobacterial NADPH-ferredoxin reductase. We also measured the kinetic parameters of cytochrome b5 reductase for cytochrome b5. Our studies are the first to report the purification and detailed biochemical characterization of the plant cytochrome b5 and cytochrome b5 reductase from the bacterial overexpression system.
Assuntos
Arabidopsis , Citocromo-B(5) Redutase , Citocromos b5 , Arabidopsis/genética , Arabidopsis/metabolismo , Domínio Catalítico , Citocromos b5/genética , Citocromos b5/metabolismo , Detergentes , Retículo Endoplasmático/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , EstirenosRESUMO
The electron-transfer capabilities of cytochrome b5 reductase (Cyt b5R) and NADPH supply have been shown to be critical factors in microbial fatty acid synthesis. Unfortunately, Cyt b5R substrate specificity is limited to the coenzyme NADH. In this study, we discovered that a novel Cyt b5R from Mortierella alpina (MaCytb5RII) displays affinity for NADPH and NADH. The enzymatic characteristics of high-purity MaCytb5RII were determined with the Km,NADPH and Km,NADH being 0.42 and 0.07 mM, respectively. MaCytb5RII shows high specific activity at 4 °C and pH 9.0. We anchored the residues that interacted with the coenzymes using the homology models of MaCytb5Rs docking NAD(P)H and FAD. The enzyme activity analysis of the purified mutants MaCytb5RII[S230N], MaCytb5RII[Y242F], and MaCytb5RII[S272A] revealed that Ser230 is essential for MaCytb5RII to have dual NAD(P)H dependence, whereas Tyr242 influences MaCytb5RII's NADPH affinity and Ala272 greatly decreases MaCytb5RII's NADH affinity.
Assuntos
Citocromo-B(5) Redutase , NAD , Citocromo-B(5) Redutase/química , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Citocromos b , Cinética , Mortierella , NAD/metabolismo , NADPRESUMO
A 6-month-old mixed breed dog was referred for evaluation of a potential disorder of sex development (DSD) and lower than expected energy level. Genitourinary examination revealed ambiguous external genitalia, hypospadias, and a subtle pouch of skin that resembled an empty scrotum. Corrective surgery was planned and subsequently aborted after cyanosis was identified preoperatively and an arterial blood gas analysis by co-oximetry identified increased methemoglobin (MetHb) concentration (35%, normal <2%) with normal arterial oxygen tension. Ensuing investigations confirmed hereditary methemoglobinemia caused by cytochrome b5 reductase (CYB5R) deficiency via molecular genetic (Arg219Pro homozygous variant in CYB5R3 gene) and biochemical (cytochrome b5 reductase enzyme activity of 8% [normal, 100% activity] testing. Karyotyping and molecular analysis of sex chromosomes revealed the dog was genetically female with a normal female karyotype (78,XX), and was negative for the Y-linked SRY gene and positive for the X-linked androgen receptor gene. Methylene blue (MB, 3.3 mg/kg per os [PO] q24 h) was administered and the MetHb concentration decreased to 9% within 14 days. Urogenital revision surgery proceeded without complication and the dog was maintained on MB (3-4 mg/kg PO q24 h) long-term without adverse effects. This is the first report to describe the use of PO MB to decrease MetHb concentrations in a dog with CYB5R deficiency in preparation for anesthesia and highlights its potential as a viable alternative to the intravenous formulation for elective procedures. In addition, this report describes the clinical, molecular, imaging, surgical, and macroscopic and microscopic pathological features of a dog with SRY-negative, 78,XX testicular DSD.
Assuntos
Doenças do Cão , Metemoglobinemia , Animais , Citocromo-B(5) Redutase/deficiência , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/uso terapêutico , Doenças do Cão/tratamento farmacológico , Doenças do Cão/genética , Doenças do Cão/cirurgia , Cães , Feminino , Masculino , Metemoglobinemia/diagnóstico , Metemoglobinemia/tratamento farmacológico , Metemoglobinemia/genética , Metemoglobinemia/veterinária , Azul de Metileno/uso terapêutico , Desenvolvimento SexualRESUMO
Detailed below is a very illustrative case of a rare pathology of recessive congenital methemoglobinemia. The patient, a newborn female, was homozygous for c.535G > A, p.(Ala179Thr) a pathogenic variant in the CYB5R3 gene. The reported population frequency of the allele is 0.853%, demonstrating why it is remarkable to find both parents are heterozygous carriers without consanguinity. A brief review of previously published cases is also presented.
Assuntos
Citocromo-B(5) Redutase , Metemoglobinemia , Cianose/genética , Citocromo-B(5) Redutase/genética , Feminino , Frequência do Gene , Homozigoto , Humanos , Recém-Nascido , Metemoglobinemia/complicações , Metemoglobinemia/congênito , Metemoglobinemia/genéticaRESUMO
BACKGROUND: Methemoglobin is the reduced form of haemoglobin that is normally found in the blood in levels < 1%. Methemoglobinemia can occur as a congenital or acquired disease. Two types of recessive congenital methaemoglobinemia (RCM) are caused by the NADH-dependent cytochrome b5 reductase enzyme deficiency of the CYB5R3 gene. RCM-I is characterized by higher methaemoglobin levels (> 2 g/dL), causing only cyanosis, whereas RCM-II is associated with cyanosis with neurological impairment. METHODS: Routine haematological investigations were done by standard method. The methaemoglobin level was evaluated by the potassium ferricyanide assay. NADH-cytochrome b5 reductase (cytb5r) enzyme activities were measured by standard methods, and molecular analysis was performed by polymerase chain reaction (PCR) followed by DNA sequencing. The interpretation of mutation effect and the molecular modeling were performed by using specific software DEEP VIEW SWISS-PDB VIEWER and Pymol molecular graphics program. RESULTS: The present study discovered three novel homozygous pathogenic variants of CYB5R3 causing RCM I and II in four unrelated Indian patients. In patient-1 and patient-2 of RCM type I caused due to novel c.175C>T (p.Arg59Cys) and other reported c.469T>C (p.Phe157Ser) missense pathogenic variants respectively, whereas patient-3 and patient-4 presented with the RCM type II are related to developmental delay with cyanosis since birth due to a novel homozygous (g.25679_25679delA) splice-site deletion and novel homozygous c.824_825insC (p.Pro278ThrfsTer367) single nucleotide insertion. The CYB5R3 transcript levels were estimated by qRT-PCR in the splice-site deletion, which was 0.33fold of normal healthy control. The insertion of nucleotide C resulted in a frameshift of termination codon are associated with neurological impairment. CONCLUSIONS: Molecular diagnosis of RCM can help to conduct genetic counselling for novel mutations and, subsequently, prenatal diagnosis of high-risk genetic disorders.