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1.
Zhonghua Nei Ke Za Zhi ; 58(7): 520-524, 2019 Jul 01.
Artigo em Chinês | MEDLINE | ID: mdl-31269569

RESUMO

Objective: To analyse the clinical characteristics of patients with acute intermittent porphyria (AIP) in order to improve the understanding and treatment. Methods: Patients diagnosed as AIP and admitted to the First Affiliated Hospital of Zhengzhou University were retrospectively enrolled from January 2008 to July 2018. Data of clinical manifestations, causes, laboratory data, treatment and clinical outcome were recorded. Results: Among the 50 patients, 41 patients (82%) were aged 20 to 40. The ratio of male and female was 1∶1.8. The most common symptoms were abdominal pain (94.0%), nausea, vomiting (72.0%) and constipation (42.0%). Neuropsychiatric disorders were seen in 72.0% patients, and 30.0% of the patients had dark-coloured urine. Precipitating factors included infections, menstruation, starvation, drugs, alcohol consumption, mental stimulation and so on. Laboratory tests were abnormal for urinary porphobilinogen, liver function, hyponatremia, anaemia and so on. Various mutations of hydroxymethylbilane synthase (HMBS) genes were detected in 16 patients. Management strategies included removal of risk factors, administration of glycogen and symptomatic treatment during acute episode. Most patients were discharged with improved conditions. Conclusions: The clinical manifestations of acute intermittent porphyria are complex and diverse. Misdiagnoses or malpractice may be fatal. It is critical to emphasize on its early diagnosis and treatment.


Assuntos
Heme/genética , Hidroximetilbilano Sintase/genética , Porfiria Aguda Intermitente/diagnóstico , Dor Abdominal/etiologia , Adulto , Feminino , Heme/metabolismo , Humanos , Hidroximetilbilano Sintase/sangue , Masculino , Mutação , Porfiria Aguda Intermitente/genética , Estudos Retrospectivos , Adulto Jovem
2.
PLoS Negl Trop Dis ; 13(4): e0007287, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30986216

RESUMO

Mosquitoes act as vectors of numerous pathogens that cause human diseases. Dengue virus (DENV) transmitted by mosquito, Aedes aegypti, is responsible for dengue fever epidemics worldwide with a serious impact on human health. Currently, disease control mainly relies on vector targeted intervention strategies. Therefore, it is imperative to understand the molecular mechanisms underlying the innate immune response of mosquitoes against pathogens. In the present study, the expression profiles of immunity-related genes in the midgut responding to DENV infection by feeding were analyzed by transcriptome and quantitative real-time PCR. The level of Antimicrobial peptides (AMPs) increased seven days post-infection (d.p.i.), which could be induced by the Toll immune pathway. The expression of reactive oxygen species (ROS) genes, including antioxidant genes, such as HPX7, HPX8A, HPX8B, HPX8C were induced at one d.p.i. and peaked again at ten d.p.i. in the midgut. Interestingly, down-regulation of the antioxidant gene HPX8C by RNA interference led to reduction in the virus titer in the mosquito, probably due to the elevated levels of ROS. Application of a ROS inhibitor and scavenger molecules further established the role of oxygen free radicals in the modulation of the immune response to DENV infection. Overall, our comparative transcriptome analyses provide valuable information about the regulation of immunity related genes in the transmission vector in response to DENV infection. It further allows us to identify novel molecular mechanisms underlying the host-virus interaction, which might aid in the development of novel strategies to control mosquito-borne diseases.


Assuntos
Aedes/genética , Aedes/imunologia , Imunidade Inata , Peroxidase/genética , Aedes/virologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Dengue/imunologia , Vírus da Dengue , Sistema Digestório/imunologia , Sistema Digestório/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Heme/genética , Heme/imunologia , Interações entre Hospedeiro e Microrganismos , Camundongos , Peroxidase/imunologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Receptores Toll-Like/genética
3.
Plant Physiol Biochem ; 137: 14-24, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30710795

RESUMO

Glutamyl-tRNA reductase1 (HEMA1) and ferrochelatase1 (FC1) are both expressed in response to salt stress in the biosynthetic pathway of tetrapyrroles. Peanut (Arachis hypogaea L.) HEMA1 and FC1 were isolated by RT-PCR. The amino acid sequence encoded by the two genes showed high similarity with that in other plant species. The AhFC1 fusion protein was verified to function in chloroplast using Arabidopsis mesophyll protoplast. Sense and wild-type (WT) tobaccos were used to further study the physiological effects of AhHEMA1 and AhFC1. Compared with WT, the Heme contents and germination rate were higher in AhFC1 overexpressing plants under salt stress. Meanwhile, overexpressing AhHEMA1 also led to higher ALA and chlorophyll contents and multiple physiological changes under salt stress, such as higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), lower contents of reactive oxygen species (ROS) and slighter membrane damage. In addition, the activities of CAT, POD and APX in the AhFC1 overexpressing plants were significantly higher than that in WT lines under salt stress, but the activity of SOD between the WT plants and the transgenic plants did not exhibit significant differences. These results suggested that, peanut can enhance resistance to salt stress by improving the biosynthesis of tetrapyrrole biosynthetic.


Assuntos
Arachis/genética , Proteínas de Plantas/genética , Estresse Salino/genética , Tabaco/genética , Ácido Aminolevulínico/metabolismo , Membrana Celular/metabolismo , Clorofila/genética , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Heme/biossíntese , Heme/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino/fisiologia , Plântula/genética , Plântula/metabolismo , Tetrapirróis/genética , Tetrapirróis/metabolismo , Tabaco/fisiologia
4.
Proc Natl Acad Sci U S A ; 116(9): 3425-3430, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755526

RESUMO

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.


Assuntos
Grupo dos Citocromos c/genética , Transporte de Elétrons/genética , Modelos Moleculares , Shewanella/genética , Sequência de Aminoácidos/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos c/química , Citocromos/química , Citocromos/genética , Elétrons , Heme/química , Heme/genética , Oxirredução , Shewanella/química , Shewanella/patogenicidade
5.
PLoS Genet ; 15(1): e1007944, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30695063

RESUMO

Heme-containing peroxidases are important components of innate immunity. Many of them functionally associate with NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes by using the hydrogen peroxide they generate in downstream reactions. Caenorhabditis elegans encodes for several heme peroxidases, and in a previous study we identified the ShkT-containing peroxidase, SKPO-1, as necessary for pathogen resistance. Here, we demonstrated that another peroxidase, HPX-2 (Heme-PeroXidase 2), is required for resistance against some, but not all pathogens. Tissue specific RNA interference (RNAi) revealed that HPX-2 functionally localizes to the hypodermis of the worm. In congruence with this observation, hpx-2 mutant animals possessed a weaker cuticle structure, indicated by higher permeability to a DNA dye, but exhibited no obvious morphological defects. In addition, fluorescent labeling of HPX-2 revealed its expression in the pharynx, an organ in which BLI-3 is also present. Interestingly, loss of HPX-2 increased intestinal colonization of E. faecalis, suggesting its role in the pharynx may limit intestinal colonization. Moreover, disruption of a catalytic residue in the peroxidase domain of HPX-2 resulted in decreased survival on E. faecalis, indicating its peroxidase activity is required for pathogen resistance. Finally, RNA-seq analysis of an hpx-2 mutant revealed changes in genes encoding for cuticle structural components under the non-pathogenic conditions. Under pathogenic conditions, genes involved in infection response were differentially regulated to a greater degree, likely due to increased microbial burden. In conclusion, the characterization of the heme-peroxidase, HPX-2, revealed that it contributes to C. elegans pathogen resistance through a role in generating cuticle material in the hypodermis and pharynx.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Imunidade Inata/genética , Oxirredutases/genética , Peroxidase/genética , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/microbiologia , Enterococcus faecalis/patogenicidade , Heme/genética , Peróxido de Hidrogênio/química , Oxirredução , Faringe/enzimologia , Faringe/microbiologia , Interferência de RNA , Homologia de Sequência de Aminoácidos
6.
Trends Parasitol ; 35(3): 213-225, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30686614

RESUMO

Traditionally, host haem has been recognized as a cytotoxic molecule that parasites need to eliminate or detoxify in order to survive. However, recent evidence indicates that some lineages of parasites have lost genes that encode enzymes involved specifically in endogenous haem biosynthesis. Such lineages thus need to acquire and utilize haem originating from their host animal, making it an indispensable molecule for their survival and reproduction. In multicellular parasites, host haem needs to be systemically distributed throughout their bodies to meet the haem demands in all cell and tissue types. Host haem also gets deposited in parasite eggs, enabling embryogenesis and reproduction. Clearly, a better understanding of haem biology in multicellular parasites should elucidate organismal adaptations to obligatory blood-feeding.


Assuntos
Heme/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Adaptação Fisiológica , Animais , Heme/biossíntese , Heme/genética
7.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30622148

RESUMO

Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.


Assuntos
Heme/genética , Hemeproteínas/genética , Complexos Multiproteicos/genética , Peptídeos/genética , Sequência de Aminoácidos , Bases de Dados Genéticas , Heme/metabolismo , Hemeproteínas/metabolismo , Humanos , Complexos Multiproteicos/química , Peptídeos/química , Ligação Proteica/genética
8.
Int J Mol Sci ; 19(7)2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29958424

RESUMO

Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme, 5-aminolevulinate synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from “gain-of-function” ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer.


Assuntos
5-Aminolevulinato Sintetase/química , Anemia Sideroblástica/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , 5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/patologia , Animais , Domínio Catalítico , Biologia Computacional , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Heme/biossíntese , Heme/genética , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutação/genética , Multimerização Proteica/genética
9.
Biochemistry ; 57(19): 2747-2755, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29658696

RESUMO

Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Heme/química , Hemeproteínas/química , Receptores de Superfície Celular/química , Proteínas Recombinantes/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/genética , Heme/genética , Hemeproteínas/genética , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Análise Espectral Raman
10.
Mol Genet Genomic Med ; 6(3): 446-451, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29500860

RESUMO

BACKGROUND: Fowler syndrome is a rare autosomal recessive disorder characterized by hydranencephaly-hydrocephaly and multiple pterygium due to fetal akinesia. To date, around 45 cases from 27 families have been reported, and the pathogenic bi-allelic mutations in FLVCR2 gene described in 15 families. The pathogenesis of this condition has not been fully elucidated so far. METHODS: We report on an additional family with two affected fetuses carrying a novel homozygous mutation in FLVCR2 gene, and describe the impact of known mutants on the protein structural and functional impairment. RESULTS: The present report confirms the genetic homogeneity of Fowler syndrome and describes a new FLVCR2 mutation affecting the protein function. The structural analysis of the present and previously published FLVCR2 mutations supports the hypothesis of a reduced heme import as the underlying disease's mechanism due to the stabilization of the occluded conformation or a protein misfolding. CONCLUSION: Our data suggest the hypothesis of heme deficiency as the major pathogenic mechanism of Fowler syndrome.


Assuntos
Hidranencefalia/genética , Proteínas de Membrana Transportadoras/genética , Receptores Virais/genética , Alelos , Sequência de Aminoácidos/genética , Feto/patologia , Heme/genética , Heme/metabolismo , Humanos , Hidranencefalia/fisiopatologia , Hidrocefalia/genética , Proteínas de Membrana Transportadoras/fisiologia , Mutação , Receptores Virais/fisiologia , Doenças Vasculares/genética
11.
J Biol Chem ; 293(5): 1850-1864, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29222330

RESUMO

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the ß1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Heme/química , Mutação de Sentido Incorreto , Guanilil Ciclase Solúvel/química , Substituição de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Heme/genética , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Guanilil Ciclase Solúvel/genética
12.
Biochemistry ; 56(46): 6111-6124, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29083920

RESUMO

Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. The Y48H variant arises from one such mutation and is found in the 40-57 Ω-loop, the lowest-unfolding free energy substructure of the cytochrome c fold. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes compared to the wild-type structure, with the axial Met80 ligand coordinated to the heme iron. Despite this, the intrinsic peroxidase activity is enhanced, implying that a pentacoordinate heme state is more prevalent in the Y48H variant, corroborated through determination of a Met80 "off rate" of >125 s-1 compared to a rate of ∼6 s-1 for the wild-type protein. Heteronuclear nuclear magnetic resonance measurements with the oxidized Y48H variant reveal heightened dynamics in the 40-57 Ω-loop and the Met80-containing 71-85 Ω-loop relative to the wild-type protein, illustrating communication between these substructures. Placed into context with the G41S cytochrome c variant, also implicated in thrombocytopenia, a dynamic picture associated with this disease relative to cytochrome c is emerging whereby increasing dynamics in substructures of the cytochrome c fold serve to facilitate an increased population of the peroxidatic pentacoordinate heme state in the following order: wild type < G41S < Y48H.


Assuntos
Citocromos c/genética , Citocromos c/metabolismo , Mutação Puntual , Cristalografia por Raios X , Citocromos c/química , Estabilidade Enzimática , Heme/química , Heme/genética , Heme/metabolismo , Humanos , Simulação de Dinâmica Molecular , Oxirredução , Peroxidase/química , Peroxidase/genética , Peroxidase/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
13.
J Biol Chem ; 292(41): 16942-16954, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28830930

RESUMO

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1, which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level.


Assuntos
Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Heme/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Glucose/metabolismo , Heme/genética , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
14.
Biochemistry ; 56(34): 4525-4538, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28762722

RESUMO

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in Escherichia coli, fully reconstituted to the respective heme b proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme b and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Heme/metabolismo , Peroxidase/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Heme/química , Heme/genética , Ligantes , Peroxidase/química , Peroxidase/genética
15.
Biotechnol Adv ; 35(6): 815-831, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28624475

RESUMO

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.


Assuntos
Biotransformação , Lacase/química , Oxirredutases/química , Dinitrocresóis/química , Fungos/química , Fungos/enzimologia , Heme/química , Heme/genética , Lacase/genética , Lignina/química , Lignina/genética , Oxirredução , Oxirredutases/classificação , Oxirredutases/genética , Peroxidases/química , Peroxidases/genética
16.
Trends Parasitol ; 33(8): 583-586, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28495484

RESUMO

The malaria parasite has a functional heme-biosynthetic pathway, although it can access host hemoglobin-heme. The heme pathway is dispensable for blood stages, but essential in the mosquito stages which do not acquire hemoglobin-heme. We propose that the blood stage parasites maintain a dynamic heme pool through multiple back-up mechanisms.


Assuntos
Heme/biossíntese , Interações Hospedeiro-Patógeno , Plasmodium falciparum/metabolismo , Animais , Culicidae/parasitologia , Heme/genética , Heme/metabolismo , Humanos , Estágios do Ciclo de Vida/fisiologia , Plasmodium falciparum/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-28540258

RESUMO

Coxiella burnetii, the etiologic agent of acute Q fever and chronic endocarditis, has a unique biphasic life cycle, which includes a metabolically active intracellular form that occupies a large lysosome-derived acidic vacuole. C. burnetii is the only bacterium known to thrive within such an hostile intracellular niche, and this ability is fundamental to its pathogenicity; however, very little is known about genes that facilitate Coxiella's intracellular growth. Recent studies indicate that C. burnetii evolved from a tick-associated ancestor and that the metabolic capabilities of C. burnetii are different from that of Coxiella-like bacteria found in ticks. Horizontally acquired genes that allow C. burnetii to infect and grow within mammalian cells likely facilitated the host shift; however, because of its obligate intracellular replication, C. burnetii would have lost most genes that have been rendered redundant due to the availability of metabolites within the host cell. Based on these observations, we reasoned that horizontally derived biosynthetic genes that have been retained in the reduced genome of C. burnetii are ideal candidates to begin to uncover its intracellular metabolic requirements. Our analyses identified a large number of putative foreign-origin genes in C. burnetii, including tRNAGlu2 that is potentially required for heme biosynthesis, and genes involved in the production of lipopolysaccharide-a virulence factor, and of critical metabolites such as fatty acids and biotin. In comparison to wild-type C. burnetii, a strain that lacks tRNAGlu2 exhibited reduced growth, indicating its importance to Coxiella's physiology. Additionally, by using chemical agents that block heme and biotin biosyntheses, we show that these pathways are promising targets for the development of new anti-Coxiella therapies.


Assuntos
Biotina/biossíntese , Coxiella burnetii/genética , Coxiella burnetii/metabolismo , Transferência Genética Horizontal , Genes Bacterianos/genética , Biotina/genética , Coxiella burnetii/crescimento & desenvolvimento , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Ácido Glutâmico/biossíntese , Ácido Glutâmico/genética , Heme/biossíntese , Heme/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Redes e Vias Metabólicas/genética , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/genética , Proteínas Virais/genética
18.
J Biol Chem ; 292(27): 11230-11242, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28533430

RESUMO

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.


Assuntos
Citocromo P-450 CYP4A/química , Ditiotreitol/química , Heme/química , Peróxido de Hidrogênio/química , Animais , Catálise , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Ditiotreitol/metabolismo , Heme/genética , Heme/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/genética , Rim/enzimologia , Fígado/enzimologia , Camundongos , Camundongos Transgênicos , Oxirredução , Ratos
19.
J Biol Chem ; 292(22): 9088-9103, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28377506

RESUMO

Hydroxyurea (HU) has a long history of clinical and scientific use as an antiviral, antibacterial, and antitumor agent. It inhibits ribonucleotide reductase and reversibly arrests cells in S phase. However, high concentrations or prolonged treatment with low doses of HU can cause cell lethality. Although the cytotoxicity of HU may significantly contribute to its therapeutic effects, the underlying mechanisms remain poorly understood. We have previously shown that HU can induce cytokinesis arrest in the erg11-1 mutant of fission yeast, which has a partial defect in the biosynthesis of fungal membrane sterol ergosterol. Here, we report the identification of a new mutant in heme biosynthesis, hem13-1, that is hypersensitive to HU. We found that the HU hypersensitivity of the hem13-1 mutant is caused by oxidative stress and not by replication stress or a defect in cellular response to replication stress. The mutation is hypomorphic and causes heme deficiency, which likely sensitizes the cells to the HU-induced oxidative stress. Because the heme biosynthesis pathway is highly conserved in eukaryotes, this finding, as we show in our separate report, may help to expand the therapeutic spectrum of HU to additional pathological conditions.


Assuntos
Heme/biossíntese , Hidroxiureia/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Heme/genética , Mutação , Estresse Oxidativo/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
20.
J Clin Invest ; 127(5): 1786-1797, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28375153

RESUMO

Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC-binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.


Assuntos
Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Heme/biossíntese , Ferro/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Anemia/genética , Anemia/metabolismo , Animais , Transporte Biológico Ativo/genética , Células CHO , Proteínas de Transporte/genética , Cricetinae , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Heme/genética , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Coativadores de Receptor Nuclear/genética
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