RESUMO
The present study evaluated the role of heme oxygenase 1 (HO-1)/carbon monoxide (CO) pathway in the cholera toxin-induced diarrhea and its possible action mechanism. The pharmacological modulation with CORM-2 (a CO donor) or Hemin (a HO-1 inducer) decreased the intestinal fluid secretion and Cl- efflux, altered by cholera toxin. In contrast, ZnPP (a HO-1 inhibitor) reversed the antisecretory effect of Hemin and potentiated cholera toxin-induced intestinal secretion. Moreover, CORM-2 also prevented the alteration of intestinal epithelial architecture and local vascular permeability promoted by cholera toxin. The intestinal absorption was not altered by any of the pharmacological modulators. Cholera toxin inoculation also increased HO-1 immunoreactivity and bilirubin levels, a possible protective physiological response. Finally, using fluorometric technique, ELISA assay and molecular docking simulations, we show evidence that CO directly interacts with cholera toxin, forming a complex that affects its binding to GM1 receptor, which help explain the antisecretory effect. Thus, CO is an essential molecule for protection against choleric diarrhea and suggests its use as a possible therapeutic tool.
Assuntos
Monóxido de Carbono , Toxina da Cólera , Humanos , Toxina da Cólera/toxicidade , Monóxido de Carbono/metabolismo , Hemina , Simulação de Acoplamento Molecular , Heme Oxigenase-1/metabolismo , Diarreia/induzido quimicamente , Diarreia/tratamento farmacológicoRESUMO
Iron uptake during infection is an essential pathogenicity factor of several bacteria, including Tenacibaculum dicentrarchi, an emerging pathogen for salmonid and red conger eel (Genypterus chilensis) farms in Chile. Iron-related protein families were recently found in eight T. dicentrarchi genomes, but biological studies have not yet confirmed functions. The investigation reported herein clearly demonstrated for the first time that T. dicentrarchi possesses different systems for iron acquisition-one involving the synthesis of siderophores and another allowing for the utilization of heme groups. Using 38 isolates of T. dicentrarchi and the type strain CECT 7612T , all strains grew in the presence of the chelating agent 2.2'-dipyridyl (from 50 to 150 µM) and produced siderophores on chrome azurol S plates. Furthermore, 37 of the 38 T. dicentrarchi isolates used at least four of the five iron sources (i.e. ammonium iron citrate, ferrous sulfate, iron chloride hexahydrate, haemoglobin and/or hemin) when added to iron-deficient media, although the cell yield was less when using hemin. Twelve isolates grew in the presence of hemin, and 10 of them used only 100 µM. Under iron-supplemented or iron-restricted conditions, whole cells of three isolates and the type strain showed at least one membrane protein induced in iron-limiting conditions (c.a. 37.9 kDa), regardless of the isolation host. All phenotypic results were confirmed by in-silico genomic T. dicentrarchi analysis. Future studies will aim to establish a relationship between iron uptake ability and virulence in T. dicentrarchi through in vivo assays.
Assuntos
Doenças dos Peixes , Tenacibaculum , Animais , Ferro/metabolismo , Sideróforos , Hemina/metabolismo , Doenças dos Peixes/microbiologia , Tenacibaculum/genética , PeixesRESUMO
Heme, an iron-containing prosthetic group found in many proteins, carries out diverse biological functions such as electron transfer, oxygen storage and enzymatic reactions. Hemin, the oxidised form of heme, is used to treat porphyria and also to activate heme-oxygenase (HO) which catalyses the rate-limiting step in heme degradation. Our group has previously demonstrated that hemin displays antitumor activity in breast cancer (BC). The aim of this work has been to study the effect of hemin on protein expression modifications in a BC cell line to gain insight into the molecular mechanisms of hemin antitumor activity. For this purpose, we carried out proteome analysis by Mass Spectrometry (MS) which showed that 1309 proteins were significantly increased in hemin-treated cells, including HO-1 and the proteases that regulate HO-1 function, and 921 proteins were significantly decreased. Furthermore, the MS-data analysis showed that hemin regulates the expression of heme- and iron-related proteins, adhesion and cytoskeletal proteins, cancer signal transduction proteins and enzymes involved in lipid metabolism. By biochemical and cellular studies, we further corroborated the most relevant in-silico results. Altogether, these results show the multiple physiological effects that hemin treatment displays in BC and demonstrate its potential as anticancer agent.
Assuntos
Neoplasias da Mama , Hemina , Humanos , Feminino , Hemina/farmacologia , Heme Oxigenase-1/metabolismo , Proteômica , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Ferro/metabolismoRESUMO
Background: Acute intermittent porphyria (AIP) is an uncommon metabolic disease, being the most common of the acute porphyrias. The most frequent symptom is acute abdominal pain, although can be accompanied by seizures, neuro-psychiatric alterations or symmetrical motor neuropathies, which in some patients can progress to respiratory musculature paralysis. Objective: To describe an atypical presentation of acute porphyria to be considered as differential diagnoses in abdominal pain. Clinical case: We present a case of a patient with AIP, presenting acute abdomen, seizures, later developed neuropsychiatric compromise and symmetrical motor neuropathy, and was admitted to mechanical ventilation. Due to the severity of the neurological involvement, he received hemin arginate, presenting with transient hypertransaminemia, an adverse event not previously reported. The evolution was favorable, with mechanical ventilation and hospital discharge withdrawn. Conclusions: The diagnosis of AIP should be considered in cases of acute abdominal pain associated with neurological and/or psychiatric symptoms, particularly young women. The administration of hemin is considered the standard of treatment, and even late could have beneficial effects.
Introducción: la porfiria aguda intermitente (PAI) es una enfermedad metabólica infrecuente, siendo la más común de las porfirias agudas. El síntoma más frecuente es el dolor abdominal agudo, aunque también pueden acompañarse de convulsiones, alteraciones neuro-psiquiátricas o neuropatías motoras simétricas, y que en algunos pacientes puede progresar a la parálisis de la musculatura respiratoria. El objetivo de este trabajo es describir una forma atípica de presentación de una porfiria aguda, a fin de considerar como diagnósticos diferenciales en dolor abdominal. Caso clínico: paciente con PAI, que presenta abdomen agudo, convulsiones, posteriormente compromiso neuro-psiquiátrico y neuropatía motora simétrica, ingresando a ventilación mecánica. Por la gravedad del compromiso neurológico recibió arginato de hemina, cursando con hipertransaminemia transitoria, evento adverso no reportado previamente. La evolución fue favorable, retirándosele la ventilación mecánica y el alta hospitalaria. Conclusiones: se debe considerar el diagnóstico de PAI en casos de dolor abdominal agudo asociado a síntomas neurológicos y/o psiquiátricos, particularmente en mujeres jóvenes. La administración de hemina es considerada el estándar de tratamiento, y aun en forma tardía podría tener efectos beneficiosos.
Assuntos
Porfiria Aguda Intermitente , Masculino , Humanos , Feminino , Porfiria Aguda Intermitente/complicações , Porfiria Aguda Intermitente/diagnóstico , Porfiria Aguda Intermitente/terapia , Hemina , Dor Abdominal/diagnóstico , Dor Abdominal/etiologia , Convulsões , Diagnóstico DiferencialRESUMO
Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.
Assuntos
Sinorhizobium meliloti , Simportadores , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Hemina/metabolismo , Ferro/metabolismo , Homeostase , Cobalto/metabolismo , Heme/metabolismoRESUMO
INTRODUÇÃO: A porfiria aguda intermitente (PAI) é um erro inato do metabolismo da biossíntese do heme, que acomete principalmente mulheres em idade menstrual. A doença se manifesta clinicamente com sintomas neuroviscerais agudos que podem ser esporádicos ou recorrentes, e que são potencialmente fatais. No Brasil, no âmbito do Sistema Único de Saúde (SUS), não há um Protocolo Clínico e Diretrizes Terapêuticas sobre o cuidado de Porfirias. Tendo em vista que a hemina (Panhematin®) é aprovada pela Anvisa para o tratamento dos ataques recorrentes de PAI relacionados com o ciclo menstrual em mulheres afetadas, quando o tratamento inicial com glicose é sabido ou suspeito de ser inadequado, o presente relatório foi desenvolvido com o objetivo de compreender a viabilidade da incorporação desse medicamento no SUS. PERGUNTA DE PESQUISA: O uso de hemina (Panhematin®) é eficaz, seguro e custo-efetivo para o tratamento de mulheres com ataques de PAI relacionados com o ciclo menstrual? EVIDÊNCIAS CLÍNICAS: Foi realizada uma revisão sistemática a partir de buscas nas bases de dados MEDLINE (via Pubmed); EMBASE; Cochrane Library; PEDRO; LILACS, IBECS BINACIS e BRISA/RedTESA (via BVS); OpenGrey; e ClinicalTrials.gov; considerando o tratamento de ataques agudos de PAI, em mulheres com idade igual ou maior a 16 anos, com hemina (Panhematin®). Foram estabelecidos critérios de elegibilidade abrangentes diante da escassez de evidências, mas ainda assim não foi encontrado nenhum ECR ou estudo observacional comparativo com resultados publicados. Foram incluídas três séries de casos que avaliaram pacientes com diferentes tipos de porfirias e sexos e 18 relatos de casos sobre mulheres com PAI. Em relação a eficácia, a redução da dor com o uso da hemina sugere que o medicamento contribua para a resolução de ataques porfíricos, mas a certeza dessa evidência é muito baixa. Somente 6 dos 18 relatos de casos avaliaram dor, sem que nenhum tenha avaliado o desfecho através de escalas, sejam elas validadas ou não. Esses estudos referiram melhora da dor na maioria dos casos. Quinze dos dezoito relatos de casos apresentam resultados para níveis urinários de ALA e PBG, sugerindo que a hemina contribui para a redução desses marcadores. Entretanto, deve-se levar em consideração a limitação das conclusões que podem ser tiradas a partir desse tipo de estudo. Quanto à segurança, os dados também são muito escassos. Duas séries de casos e três relatos de casos descreveram resultados para eventos adversos graves e não foram identificadas novas preocupações além daquelas descritas em bula. Todas as evidências incluídas neste relatório apresentaram risco de viés muito alto ou alto e certeza do conjunto de evidências avaliado como muito baixa, de acordo com o GRADE. Mais estudos são necessários para permitir conclusões mais robustas acerca da eficácia e, sobretudo, da segurança do medicamento. AVALIAÇÃO ECONÔMICA: Foi elaborado um modelo de custo-efetividade, baseando-se no desfecho de mortalidade, sob a perspectiva do SUS, com horizonte temporal de um ano, e incluindo apenas custos diretos (de tratamento com as terapias selecionadas e de internação). A análise foi conduzida a partir de em um modelo de árvore de decisão que projetou a relação entre custo da intervenção (hemina + glicose) e do comparador (glicose). O resultado da razão de custoefetividade incremental (RCEI) da hemina em comparação à glicose foi de R$ 1.101.525,27por vida salva, variando de R$450.511,78 a R$5.477.345,10 por vida salva na análise de sensibilidade probabilística. ANÁLISE DE IMPACTO ORÇAMENTÁRIO: Adotou-se uma perspectiva epidemiológica considerando os dados de prevalência e incidência de PAI disponíveis em literatura e aplicados ao contexto do Brasil, bem como proporção de mulheres que tinham a condição, o número de ataques e a utilização de hemina para o tratamento das crises. Foram considerados os mesmos recursos e custos associados utilizados na análise de custo-efetividade, assumindo disponibilidade da hemina no mercado de forma progressiva. A incorporação da hemina (Panhematin®), no SUS, geraria um impacto incremental de cerca de R$ 11,1 milhões no primeiro ano, atingindo quase R$ 83 milhões em cinco anos. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: não foram detectadas tecnologias para compor o esquema terapêutico de ataques de PAI relacionados com o ciclo menstrual. Vale ressaltar que o medicamento givosirana foi identificado nas buscas, entretanto, o ensaio clínico identificado no Clinicaltrials avalia o uso da givosirana no tratamento de porfirias hepáticas agudas de forma geral, sem especificar a inclusão de PAI relacionada ao ciclo menstrual. O mesmo acontece para a indicação clínica do medicamento aprovada na Anvisa (2020), FDA (2019) e EMA (2020). CONSIDERAÇÕES FINAIS: há escassez de evidências sobre a eficácia e segurança da hemina (Panhematin®) e as evidências disponíveis provém apenas de séries de casos e relatos de casos, o que torna a avaliação de eficácia e segurança um desafio. Além do delineamento dos estudos, a descrição incompleta e a avaliação subjetiva dos resultados são outras dificuldades. As limitações acabam por se estender para as análises econômicas, as quais foram baseadas em estudos que não atendiam à indicação de bula da tecnologia. A análise de custo-efetividade incremental e o impacto orçamentário mostram valores expressivos e a incorporação dela indica não colaborar para a sustentabilidade do SUS, embora a hemina seja a única alternativa farmacológica comercializada atualmente para o manejo de crises de PAI. RECOMENDAÇÃO PRELIMINAR: os membros presentes no plenário da Conitec, em sua 110ª Reunião Ordinária, realizada no dia 06 de julho de 2022, deliberaram que a matéria fosse disponibilizada em consulta pública com recomendação preliminar desfavorável à incorporação de Panhematin® para o tratamento da porfiria aguda intermitente relacionados com o ciclo menstrual. Para essa recomendação, a Conitec considerou que as evidências acerca da eficácia e segurança do medicamento são escassas e de baixa qualidade. RECOMENDAÇÃO FINAL DA CONITEC: Pelo exposto, o Plenário da Conitec, em sua 112ª Reunião Ordinária, realizada no dia 31 de agosto de 2022, deliberou por unanimidade recomendar a não incorporação da hemina para o tratamento da porfiria aguda intermitente relacionados com o ciclo menstrual. Os membros da Conitec consideraram que não houve argumentação suficiente para a mudança de entendimento acerca da recomendação preliminar, principalmente considerando as evidências para eficácia e segurança da tecnologia, inclusive após contribuição presente de especialista no assunto. Foi assinado o Registro de Deliberação nº 768/2022. DECISÃO: não incorporar, no âmbito do Sistema Único de Saúde - SUS, a hemina para o tratamento da porfiria aguda intermitente relacionada com o ciclo menstrual, conforme a Portaria nº 110, publicada no Diário Oficial da União nº 184, seção 1, página 76, em 27 de setembro de 2022.
Assuntos
Humanos , Porfiria Aguda Intermitente/tratamento farmacológico , Hemina/uso terapêutico , Ciclo Menstrual , Sistema Único de Saúde , Brasil , Análise Custo-Benefício/economiaRESUMO
Hemopexin (Hx) is a plasma glycoprotein that scavenges heme (Fe(III) protoporphyrin IX). Hx has important implications in hemolytic disorders and hemorrhagic conditions because releasing hemoglobin increases the labile heme, which is potentially toxic, thus producing oxidative stress. Therefore, Hx has been considered for therapeutic use and diagnostics. In this work, we analyzed and mapped the interaction sequences of Hx with hemin and hemoglobin. The spot-synthesis technique was used to map human hemopexin (P02790) binding to hemin and human hemoglobin. A library of 15 amino acid peptides with a 10-amino acid overlap was designed to represent the entire coding region (aa 1-462) of hemopexin and synthesized onto cellulose membranes. An in silico approach was taken to analyze the amino acid frequency in the identified interaction regions, and molecular docking was applied to assess the protein-protein interaction. Seven linear peptide sequences in Hx were identified to bind hemin (H1-H7), and five were described for Hb (Hb1-Hb5) interaction, with just two sequences shared between hemin and Hb. The amino acid composition of the identified sequences demonstrated that histidine residues are relevant for heme binding. H105, H293, H373, H400, H429, and H462 were distributed in the H1-H7 peptide sequences, but other residues may also play an important role. Molecular docking analysis demonstrated Hx's association with the ß-chain of Hb, with several hotspot amino acids that coordinated the interaction. This study provides new insights into Hx-hemin binding motifs and protein-protein interactions with Hb. The identified binding sequences and specific peptides can be used for therapeutic purposes and diagnostics as hemopexin is under investigation to treat different diseases and there is an urgent need for diagnostics using labile heme when monitoring hemolysis.
Assuntos
Hemina , Hemopexina , Compostos Férricos , Heme/metabolismo , Hemina/metabolismo , Hemoglobinas/metabolismo , Hemólise , Hemopexina/metabolismo , Histidina , Humanos , Simulação de Acoplamento MolecularRESUMO
(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.
Assuntos
COVID-19/etiologia , Hemoglobinas/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , COVID-19/sangue , Hemina/metabolismo , Hemoglobinas/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteômica , Protoporfirinas/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Estruturais Virais/ultraestrutura , Ligação Viral , Replicação ViralRESUMO
Meat diet plays a pivotal role in colorectal cancer (CRC). Hemin, a metabolite of myoglobin, produced after meat intake, has been involved in CRC initiation. The compound, 3,4-dihydroxyphenylacetic acid (3,4HPAA) is a scarcely studied microbiota-derived metabolite of the flavonoid quercetin (QUE), which exert antioxidant properties. The aim of this study was to determine the protective effect of 3,4HPAA against malignant transformation (increased cell proliferation, decreased apoptosis, DNA oxidative damage and augmented reactive oxidative species (ROS) levels) and mitochondrial dysfunction induced by hemin in normal colon epithelial cells and colon cancer cells. The effect of 3,4HPAA was assessed in comparison to its precursor, QUE and to a known CRC protective agent, sulforaphane (SFN). The results showed that both, tumor and normal cells, exposed to hemin, presented increased cell proliferation, decreased caspase 3 activity and cytochrome c release, as well as augmented production of intracellular and mitochondrial ROS. In addition, hemin decreased the mitochondrial membrane potential (MMP) and the activity of complexes I and II of the electron transport chain. These effects of hemin were prevented by the action of 3,4HPAA. The metabolite showed to be more active than QUE and slightly less active than SFN. In conclusion, 3,4HPAA administration could represent a promising strategy for preventing malignant transformation and mitochondrial dysfunction in colon epithelia induced by hemin.