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
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
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
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 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
Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.
Assuntos
Citoglobina/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Aorta/citologia , Sobrevivência Celular/efeitos dos fármacos , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Citoglobina/genética , Técnicas de Silenciamento de Genes , Camundongos , Músculo Liso Vascular/citologia , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Cytochrome b5 reductase (Cb5R) is a flavoprotein that participates in the reduction of multiple biological redox partners. Co-localization of this protein with nitric oxide sources has been observed in neurons. In addition, the generation of superoxide anion radical by Cb5R has been observed. A search for specific inhibitors of Cb5R to understand the role of this protein in these new functions has been initiated. Previous studies have shown the ability of different flavonoids to inhibit Cb5R. Anthocyanins are a subgroup of flavonoids responsible for most red and blue colors found in flowers and fruits. Although usually represented by the flavylium cation form, these species are only stable at rather acidic pH values (pH ≤ 1). At higher pH values, the flavylium cation is involved in a dynamic reaction network comprising different neutral species with the potential ability to inhibit the activities of Cb5R. This study aims to provide insights into the molecular mechanism of interaction between flavonoids and Cb5R using flavylium salts as dynamic inhibitors. The outcome of this study might lead to the design of improved specific enzyme inhibitors in the future.
Assuntos
Antocianinas , Sais , Humanos , Citocromo-B(5) Redutase/química , Citocromo-B(5) Redutase/metabolismo , Superóxidos , Flavonoides/farmacologia , CátionsRESUMO
Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the "140s loop".
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Transporte de Elétrons/genética , Elétrons , Mononucleotídeo de Flavina/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sequência de Aminoácidos , Animais , Família 2 do Citocromo P450/metabolismo , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Glicina/genética , Cinética , Microssomos/metabolismo , Mutagênese Sítio-Dirigida/métodos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Oxirredução , Ligação Proteica , Conformação Proteica , CoelhosRESUMO
NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2â¢-. The ratio of NOX4-derived H2O2 and O2â¢- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2â¢-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.
Assuntos
Citocromo-B(5) Redutase/metabolismo , Células Endoteliais , Peróxido de Hidrogênio , Animais , Células Cultivadas , Endotélio , Inflamação/genética , Camundongos , NADPH Oxidase 4/genética , NADPH Oxidases , Espécies Reativas de Oxigênio , UbiquinonaRESUMO
Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5, that could be used in binding assays at sub-µM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4â¢Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high, and that ternary P450-b 5-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT: High-affinity binding of cytochrome b 5 (b 5) (K d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b 5-NADPH-P450 reductase complex.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450 , Citocromos b5/metabolismo , Fluoresceínas/farmacocinética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ácidos Sulfônicos/farmacocinética , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo-B(5) Redutase/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/farmacocinética , Humanos , Microssomos Hepáticos/metabolismo , Ensaio Radioligante/métodosAssuntos
Coenzima A Ligases/genética , Sistema Enzimático do Citocromo P-450/genética , Ferroptose/genética , Peroxidação de Lipídeos/genética , Peróxidos Lipídicos/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Retículo Endoplasmático/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Ácidos Graxos Insaturados/metabolismo , Regulação da Expressão Gênica , Humanos , Ferro/metabolismo , Oxigênio/metabolismoRESUMO
Diabetic retinopathy (DR) is a major cause of vision loss in working and elderly populations. long non-coding RNA (LncRNA) MEG3 is thought to have some effect on DR, but the exact mechanism remains to be clarified. The expression levels of lncRNA MEG3, miR-6720-5p, and cytochrome B5 reductase 2 (CYB5R2) in human retinal microvascular endothelial cells (hRMECs) were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), transwell migration, and tube formation assays were used to determine the cell viability, migration, and tube formation ability of hRMECs, respectively. The interaction of MEG3, miR-6720-5p, and CYB5R2 was detected and explored by a luciferase assay. The expression of MEG3 and CYB5R2 was upregulated and that of miR-6720-5p was downregulated in patients with DR and hRMECs treated with high glucose. Knocking down MEG3 or CYB5R2 promoted proliferation, migration, and neovascularization in hRMECs. The intervention of miR-6720-5p reversed the effect of MEG3 knockdown on hRMEC function, and this effect was eliminated by silencing CYB5R2. Therefore, MEG3 acted as a sponge to suppress miR-6720-5p and regulate the expression of CYB5R2, thereby inhibiting DR neovascularization.
Assuntos
Citocromo-B(5) Redutase/genética , Retinopatia Diabética/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Neovascularização Patológica/genética , RNA Longo não Codificante/metabolismo , Animais , Sequência de Bases , Estudos de Casos e Controles , Citocromo-B(5) Redutase/metabolismo , Retinopatia Diabética/sangue , Feminino , Inativação Gênica , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Neovascularização Patológica/sangue , RNA Longo não Codificante/sangue , RNA Longo não Codificante/genética , Ratos Sprague-DawleyRESUMO
Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins might eventually transfer electrons to oxygen to form reactive oxygen species. Additionally, an inefficient electron transfer to redox acceptors can lead to electron uncoupling and superoxide anion formation by the reductase. Many efforts have been made to characterize the involved catalytic domains in the electron transfer from the reduced flavoprotein to its electron acceptors, such as cytochrome b5, through a detailed description of the flavin and NADH-binding sites. This information might help to understand better the processes and modifications involved in reactive oxygen formation by the cytochrome b5 reductase. Nevertheless, more than half a century since this enzyme was first purified, the one-electron transfer process toward potential electron acceptors of the reductase is still only partially understood. New advances in computational analysis of protein structures allow predicting the intramolecular protein dynamics, identifying potential functional sites, or evaluating the effects of microenvironment changes in protein structure and dynamics. We applied this approach to characterize further the roles of amino acid domains within cytochrome b5 reductase structure, part of the catalytic domain, and several sensors and structural domains involved in the interactions with cytochrome b5 and other electron acceptors. The computational analysis results allowed us to rationalize some of the available spectroscopic data regarding ligand-induced conformational changes leading to an increase in the flavin adenine dinucleotide (FAD) solvent-exposed surface, which has been previously correlated with the formation of complexes with electron acceptors.
Assuntos
Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Transporte de Elétrons/fisiologia , Flavina-Adenina Dinucleotídeo/metabolismo , HumanosRESUMO
OBJECTIVE: Cytochrome b5-reductase (CYB5R) and cytochrome P450 reductase (CYPOR) are important for cell metabolism; however, their role in thyroid hormonogenesis and carcinogenesis has not been elucidated yet. The activity of CYB5R correlates with the metastasis in breast cancer, but there are no similar studies for CYB5R and CYPOR for thyroid tumors. The aim of this study was to elucidate the activity of CYB5R and CYPOR changes in benign euthyroid and hyperthyroid neoplasms and in papillary thyroid cancer for their potential application as biomarkers for diagnosis and prognosis prediction of thyroid cancer. METHODS: Thirty-six patients with thyroid diseases participated in the study. The control euthyroid nodular goiter group included ten patients; the thyrotoxic nodular or diffuse goiter group included 14 patients; the papillary thyroid cancer T1-2N0-1M0 (PTC) group included 12 patients. The activity of CYB5R and CYPOR was assessed with lucigenin-enhanced chemiluminescence stimulated by NADH and NADPH, respectively. RESULTS: Compared to the control euthyroid nodular goiter group, activity of CYB5R and CYPOR increased ~5 and 10 times, respectively, in toxic goiter, and 15 and 30 times, respectively, in half of cases of PTC. The change in activity of CYPOR was more pronounced compared to CYB5R. Within the PTC group, the subgroups with low and high activities of microsomal reductases were identified. Microsomal reductases in follicular adenoma was 2-4-fold less active compared to the euthyroid nodular goiter and the low-activity PTC group. CONCLUSIONS: Activity of tissue microsomal reductases varies in thyroid pathology and can be considered as a promising biomarker for differential diagnostics of benign and malignant thyroid tumors.