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1.
Subcell Biochem ; 94: 251-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189303

RESUMO

There are three broad groups of oxygen-transport proteins found in the haemolymph (blood) of invertebrates, namely the hemocyanins, the hemerythrins and the globins. Both hemerythrins and extracellular globins are iron-based proteins that are understudied when compared to the copper-containing hemocyanins. Recent evidence suggests that hemerythrins and (giant) extracellular globins (and their linker chains) are more widely distributed than previously thought and may have biological functions beyond oxygen transport and storage. Herein, we review contemporary literature of these often-neglected proteins with respect to their structural configurations on formation and ancestral states.


Assuntos
Evolução Molecular , Globinas/química , Hemeritrina/química , Hemocianinas/química , Invertebrados/química , Animais
2.
Mar Genomics ; 49: 100721, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31711848

RESUMO

Since the discovery of new members of the globin superfamily such as Cytoglobin, Neuroglobin and Globin X, in addition to the most well-known members, Hemoglobin and Myoglobin, different hypotheses have been suggested about their function in vertebrates. Globins are ubiquitously found in living organisms and can carry out different functions based on their ability to bind ligands such as O2, and nitric oxide (NO) and to catalyze reactions scavenging NO or generating NO by reducing nitrite. NO is a highly diffusible molecule with a central role in signaling important for egg maturation, fertilization and early embryonic development. The globins ability to scavenge or generate NO makes these proteins ideal candidates in regulating NO homeostasis depending on the micro environment and tissue NO demands. Different amounts of various globins have been found in zebrafish eggs and developing embryos where it's unlikely that they function as respiratory proteins and instead could play a role in maintaining embryonic NO homeostasis. Here we summarize the current knowledge concerning the role of NO in adult fish in comparison to mammals and we discuss NO function during embryonic development with possible implications for globins in maintaining embryonic NO homeostasis.


Assuntos
Desenvolvimento Embrionário , Peixes/embriologia , Globinas/fisiologia , Óxido Nítrico/fisiologia , Animais , Homeostase
3.
Mar Genomics ; 49: 100724, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31735579

RESUMO

The ancient origins and functional versatility of globins make them ideal subjects for studying physiological adaptation to environmental change. Our goals in this review are to describe the evolution of the vertebrate globin gene superfamily and to explore the structure/function relationships of hemoglobin, myoglobin, neuroglobin and cytoglobin in teleost fishes. We focus on the globins of Antarctic notothenioids, emphasizing their adaptive features as inferred from comparisons with human proteins. We dedicate this review to Guido di Prisco, our co-author, colleague, friend, and husband of C.V. Ever thoughtful, creative, and enthusiastic, Guido spearheaded study of the structure, function, and evolution of the hemoglobins of polar fishes - this review is testimony to his wide-ranging contributions. Throughout his career, Guido inspired younger scientists to embrace polar biological research, and he challenged researchers of all ages to explore evolutionary adaptation in the context of global climate change. Beyond his scientific contributions, we will miss his warmth, his culture, and his great intellect. Guido has left an outstanding legacy, one that will continue to inspire us and our research.


Assuntos
Adaptação Fisiológica , Evolução Molecular , Peixes/genética , Globinas/genética , Sequência de Aminoácidos , Animais , Regiões Antárticas , Citoglobina/genética , Hemoglobinas/genética , Família Multigênica , Mioglobina/genética , Neuroglobina/genética , Sintenia
5.
BMC Genomics ; 20(1): 741, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615396

RESUMO

BACKGROUND: Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of QuantSeq 3'mRNA sequencing (QuantSeq) combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of QuantSeq using a novel specific globin blocker (GB) that is included in the library preparation step of QuantSeq. RESULTS: In data set 1, four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads compared to non-GB (NGB) samples (P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads compared to the NGB (from 56.4 to 10.1%). The second highest concentration C2, which showed very similar globin depletion rates (12%) as C1 but a better correlation of the expression of non-globin genes between NGB and GB (r = 0.98), allowed the expression of an additional 1295 non-globin genes to be detected, although 40 genes that were detected in the NGB sample (at a low level) were not present in the GB library. Concentration C2 was applied in the rest of the study. In data set 2, the distribution of the percentage of globin reads for NGB (n = 184) and GB (n = 189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB, similar to results from data set 1. Data set 3 (n = 84) revealed that the proportion of globin reads that remained in GB samples was significantly and positively correlated with the reticulocyte count in the original blood sample (P < 0.001). CONCLUSIONS: The effect of the GB on reducing the proportion of globin reads in porcine blood QuantSeq was demonstrated in three data sets. In addition to increasing the efficiency of sequencing non-globin mRNA, the GB for QuantSeq has an advantage that it does not require an additional step prior to or during library creation. Therefore, the GB is a useful tool in the quantification of whole gene expression profiles in porcine blood.


Assuntos
Perfilação da Expressão Gênica/veterinária , Globinas/antagonistas & inibidores , RNA Mensageiro/sangue , Regiões 3' não Traduzidas , Animais , Feminino , Análise de Sequência de RNA , Suínos
6.
Arch Biochem Biophys ; 673: 108079, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31445024

RESUMO

The multifunctional catalytic hemoglobin dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of EPA Priority Pollutants (4-Me-o-cresol, 4-Cl-m-cresol and pentachlorophenol) and EPA Toxic Substances Control Act compounds (o-, m-, p-cresol and 4-Cl-o-cresol). Biochemical assays (HPLC/LC-MS) indicated formation of multiple oxidation products, including the corresponding catechol, 2-methylbenzoquinone (2-MeBq), and oligomers with varying degrees of oxidation and/or dehalogenation. Using 4-Br-o-cresol as a representative substrate, labeling studies with 18O confirmed that the O-atom incorporated into the catechol was derived exclusively from H2O2, whereas the O-atom incorporated into 2-MeBq was from H2O, consistent with this single substrate being oxidized by both peroxygenase and peroxidase mechanisms, respectively. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the peroxygenase mechanism leading to catechol formation, and for Compounds I and ES in the peroxidase mechanism that yields the 2-MeBq product. The X-ray crystal structures of DHP bound with 4-F-o-cresol (1.42 Å; PDB 6ONG), 4-Cl-o-cresol (1.50 Å; PDB 6ONK), 4-Br-o-cresol (1.70 Å; PDB 6ONX), 4-NO2-o-cresol (1.80 Å; PDB 6ONZ), o-cresol (1.60 Å; PDB 6OO1), p-cresol (2.10 Å; PDB 6OO6), 4-Me-o-cresol (1.35 Å; PDB 6ONR) and pentachlorophenol (1.80 Å; PDB 6OO8) revealed substrate binding sites in the distal pocket in close proximity to the heme cofactor, consistent with both oxidation mechanisms. The findings establish cresols as a new class of substrate for DHP, demonstrate that multiple oxidation mechanisms may exist for a given substrate, and provide further evidence that different substituents can serve as functional switches between the different activities performed by dehaloperoxidase. More broadly, the results demonstrate the complexities of marine pollution where both microbial and non-microbial systems may play significant roles in the biotransformations of EPA-classified pollutants, and further reinforces that heterocyclic compounds of anthropogenic origin should be considered as environmental stressors of infaunal organisms.


Assuntos
Poluentes Ambientais/metabolismo , Globinas/metabolismo , Oxigenases de Função Mista/metabolismo , Peroxidase/metabolismo , United States Environmental Protection Agency , Biocatálise , Oxigenases de Função Mista/química , Modelos Moleculares , Peroxidase/química , Conformação Proteica , Estados Unidos
7.
Oxid Med Cell Longev ; 2019: 6315034, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354909

RESUMO

Reactive oxygen species (ROS) result from intracellular aerobic metabolism and/or extracellular stimuli. Although endogenous antioxidant systems exquisitely balance ROS production, an excess of ROS production, commonly found in diverse human degenerative pathologies including cancer, gives rise to the oxidative stress. Increased oxidative stress in cancer is related to the sustained proliferation and metabolism of cancer cells. However, cancer cells show an intrinsic higher antioxidant capacity with respect to the normal counterpart as well as an ability to cope with oxidative stress-induced cell death by establishing mechanisms of adaptation, which define a selective advantage against the adverse oxidative stress environment. The identification of survival factors and adaptive pathways, set up by cancer cells against oxidative stress, provides multiple targets for the therapeutic intervention against cancer. Neuroglobin (NGB), a globin primarily described in neurons as an oxidative stress sensor and cytoprotective factor against redox imbalance, has been recently recognized as a novel tumor-associated protein. In this review, the involvement of NGB in the cancer cell adaptation and resistance to oxidative stress will be discussed highlighting the globin role in the regulation of both the stress-induced apoptotic pathway and antioxidant systems activated by cancer cells.


Assuntos
Neoplasias/tratamento farmacológico , Neuroglobina/uso terapêutico , Morte Celular , Globinas , Humanos , Neuroglobina/farmacologia , Estresse Oxidativo , Espécies Reativas de Oxigênio
8.
Med Sci Monit ; 25: 3181-3189, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31038126

RESUMO

BACKGROUND Modern medicine has suggested exercise therapy is one of the main treatments for postoperative rehabilitation of tumors. It can influence the recovery of cancer patients by changing the body's material metabolism and energy metabolism. However, studies on metabolic changes of exercise therapy on hepatocellular carcinoma (HCC) patients after surgery are limited. The aim of this study was to explore the effect of aerobic exercise on mice after orthotopic HCC surgery by serum metabolomics test and explore the related mechanism. MATERIAL AND METHODS A total of 60 C57Bl/6 mice were used to establish an orthotopic xenograft model of H22 mouse hepatoma cells. Mice were randomly divided into 6 groups and it was found that the metabolic products of the early postoperative exercise group and sedentary group mainly included L-tryptophan, citric acid, and other energy-related metabolites. RESULTS Energy metabolites, such as succinic acid of the high-intensity exercise group were increased after surgery, whereas phospholipid metabolites, including phosphatidylethanolamine (18: 0/0: 0), were decreased. In the moderate-intensity exercise group, the change tendency was consistent, and the level of various metabolites decreased. CONCLUSIONS Thus, it is likely that aerobic exercise reduced the degree of postoperative stress responses and improved energy metabolism in mice. The underlying mechanism involves improving the tricarboxylic acid cycle, intervening in energy metabolism, reorganization caused by the tumor, reducing the abnormal increase of phospholipase activity caused by the stress of liver cancer, reducing the level of hemolytic phospholipids, thereby inhibiting mitochondrial pathway-initiated apoptosis.


Assuntos
Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/cirurgia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/cirurgia , Condicionamento Físico Animal/métodos , Alanina Transaminase/sangue , Animais , Apoptose/fisiologia , Aspartato Aminotransferases/sangue , Metabolismo Energético , Globinas/metabolismo , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Masculino , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Albumina Sérica/metabolismo
9.
PLoS Genet ; 15(4): e1008039, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30970016

RESUMO

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.


Assuntos
Sarcoma Experimental/etiologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Nuclear Ligada ao X/deficiência , Proteína Nuclear Ligada ao X/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Carcinogênese/genética , Carcinogênese/metabolismo , Modelos Animais de Doenças , Eritropoese , Feminino , Técnicas de Inativação de Genes , Globinas/genética , Humanos , Mutação com Perda de Função , Masculino , Neurofibromina 1/deficiência , Neurofibromina 1/genética , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Homeostase do Telômero/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
10.
Int Arch Occup Environ Health ; 92(6): 873-881, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30955093

RESUMO

OBJECTIVES: Urinary excretion of 2,5-hexanedione is currently used to estimate the exposure levels of hexane occurring to an individual during the previous work shift. However, because hexane exposures and urinary 2,5-hexanedione levels can vary considerably from day to day, and subchronic to chronic exposures to hexane are required to produce neuropathy, this biomarker may not accurately reflect the risk of an individual for developing hexane neuropathy. This investigation examines the potential of hexane-derived pyrrole adducts produced on globin and plasma proteins as markers for integrating cumulative exposures. Because the pyrrole markers incorporate bioactivation of hexane to 2,5-hexandione and the initial step of protein adduction involved in hexane-induced neuropathy, they potentially can serve as biomarkers of effect through reflecting pathogenetic events within the nervous system. Additionally, pyrrole formation is an irreversible reaction suggesting that hexane-derived protein pyrroles can be used to assess cumulative exposures to provide a better characterization of individual susceptibilities. METHODS: To examine the utility of the proposed markers, blood samples were obtained from eleven workers who used hexane for granulating metal powders in a slurry to produce metal machining die tools and four non-exposed volunteers. Globin and plasma were isolated, and the proteins were digested using pepsin, reacted with Ehrlich's reagent and the level of pyrrole adducts were determined by absorbance at 530 nm. To determine the dose-response curve and dynamic range of the assay, erythrocytes were incubated with a range of 2,5-hexanedione concentrations and the net absorbance at 530 nm of isolated globin was measured. RESULTS: Pyrrole was detected in both the globin and plasma samples of the workers exposed to hexane and the levels of pyrroles in plasma were positively correlated with the levels of pyrroles in globin for most of the workers. CONCLUSIONS: This investigation demonstrates that detectable levels of hexane-derived protein pyrrole adducts are produced on peripheral proteins following occupational exposures to hexane and supports the utility of measuring pyrroles for integrating cumulative exposures to hexane.


Assuntos
Globinas/metabolismo , Hexanos/metabolismo , Plasma/química , Pirróis/sangue , Biomarcadores/sangue , Globinas/química , Humanos , Exposição Ocupacional/efeitos adversos , Pirróis/metabolismo
11.
Mol Biol Evol ; 36(6): 1134-1147, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30828717

RESUMO

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.


Assuntos
Adaptação Biológica , Evolução Molecular , Globinas/genética , Pseudogenes , Sirênios/genética , Animais , Conversão Gênica , Globinas/metabolismo , Masculino , Família Multigênica , Proteínas Mutantes Quiméricas , Oxigênio/metabolismo , Seleção Genética , Sirênios/metabolismo
12.
Blood ; 133(12): 1358-1370, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30700418

RESUMO

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.


Assuntos
Anemia de Diamond-Blackfan/patologia , Diferenciação Celular , Células Eritroides/patologia , Fator de Transcrição GATA1/metabolismo , Globinas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Heme/metabolismo , Anemia de Diamond-Blackfan/metabolismo , Proliferação de Células , Células Cultivadas , Células Eritroides/metabolismo , Feminino , Seguimentos , Haploinsuficiência , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Fenótipo , Prognóstico , RNA Interferente Pequeno , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
13.
Metab Brain Dis ; 34(3): 705-713, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30701417

RESUMO

Silver nanoparticles (AgNPs) are clusters of silver atoms with diameters that range from 1 to 100 nm. Due to the various shapes and large surface areas, AgNPs have been employed in the food and textile industries and medical fields. Therefore, because of the widespread use of these compounds, the aim of this study was to evaluate the effect of AgNP exposure on the gene and protein expression levels of Neuroglobin (Ngb) and Cytoglobin (Cygb), in the rat cortex, hippocampus and cerebellum. Post-natal day (PND) 21 male Wistar rats were randomly divided into three groups. One group received 15 µg/kg body weight of AgNP by gavage another group received 30 µg/kg and the control group that received saline, from PND23 to PND58. On PND102 the animals were euthanized and the cortex, hippocampus and cerebellum were isolated and evaluated for gene and protein expression levels of Nbg and Cygb. The results demonstrated that the 30 µg/kg AgNP group displayed increased gene and protein expression of Cygb in the cortex. In the Hippocampus, AgNP exposure did not modulate gene or protein expression levels of Ngb and Cygb. In cerebellum the Ngb gene and protein expression was increased with both doses of AgNP. AgNP exposure during prepubescence can modulate the gene and protein expression levels of Ngb and Cygb in adulthood. Furthermore, the observed modulation was specific to the cerebellum, and cortex, and was dose dependent.


Assuntos
Citoglobina/metabolismo , Nanopartículas Metálicas/toxicidade , Neuroglobina/metabolismo , Prata/toxicidade , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Globinas/efeitos dos fármacos , Globinas/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Ratos Wistar
14.
Mol Biol Rep ; 46(2): 2101-2110, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30729391

RESUMO

Truncated globins are 20-40 amino acids shorter than full length globins. Till date, globins have been characterized predominantly from bacteria involved in pathogenicity, nitrogen fixation and photosynthesis, where they are implicated in bacterial virulence within the host, protection of nitrogenase from oxygen inactivation and prevention of oxidative damage to the photosynthetic machinery respectively. Myxococcus xanthus, the model myxobacterium, is an obligate aerobe with a multicellular stage in its life cycle where cells encounter oxygen limitation. This work was undertaken to investigate the potential role of the truncated globin in M. xanthus. To examine the role of globins in this unique group of bacteria, the gene coding for a putative truncated globin (HbO) was identified in the genome of M. xanthus DK 1622. The sequence analysis by bioinformatics approaches revealed that HbO from M. xanthus (Mx-HbO) likely adopts a 2-on-2 alpha helical fold of the truncated globins. The gene coding for Mx-HbO was cloned and its expression in E. coli imparted reddish tinge to the cells. The spectral analysis confirmed it to be a functional globin. The expression of Mx-HbO in the heterologous host improved its growth, resulting in the attainment of higher cell density in culture. The transcript of Mx-hbO was induced threefold in the host cells when grown under low aeration condition as compared to the cells grown under high aeration condition. In M. xanthus, an obligate aerobe, where cell growth accompanies swarming, there is a higher density of cells in the middle of the swarm. Our results suggest that Mx-HbO is a functional globin and could facilitate the growth of cells facing oxygen deprivation, the condition prevailing in the middle of the swarm.


Assuntos
Globinas/genética , Myxococcus xanthus/genética , Hemoglobinas Truncadas/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Simulação por Computador , Escherichia coli/genética , Globinas/metabolismo , Myxococcus xanthus/metabolismo , Transcrição Genética/genética , Hemoglobinas Truncadas/metabolismo
15.
PLoS One ; 14(2): e0212492, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30768627

RESUMO

BACKGROUND: B-cell lymphoma/leukaemia 11A (BCL11A) is a C2H2-type zinc-finger transcription factor protein that is a critical modulator of haemoglobin switching and suppresses the production of foetal haemoglobin. Variation in the BCL11A gene ameliorates the severity of sickle cell disease (SCD) and ß-thalassemia (ß-thal). The BCL11A gene is located on chromosome 2p16.1 and encodes an 835-amino acid protein. METHOD: Using state-of-the-art in silico tools, this study examined the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) that disrupt the BCL11A protein and mediate foetal-to-adult globin switching. A total of 11,463 SNPs were retrieved from the Single Nucleotide Polymorphism database (dbSNP). These included 799 in the 5' untranslated region (UTR), 486 in the 3' UTR, and 266 non-synonymous, 189 coding synonymous, six nonsense, and six stop-gained SNPs. RESULTS AND DISCUSSION: In silico tools (SIFT, SNAP, PolyPhen-2, PANTHER, I-Mutant, PROVEAN, SNPs&GO, mCSM, and PhD-SNP) predicted the five most-deleterious nsSNPs: rs61742690, rs62142605, rs17028351, rs115666026, and rs74987258. Molecular dynamic simulation and homology modelling of the mutated proteins (S783N, D643N, G451S, K670R, and M313L) of the most deleterious nsSNPs revealed their functional and structural impact. nsSNP rs61742690 was predicted to be the most deleterious, as supported by eight of the nine in silico tools. CONCLUSIONS: Complete failure in the protein-protein interactions with functional partners (KLF1 and others) and significant changes (±100% variation) in the interface energy revealed that rs61742690 (S783N) in the zinc-finger domain is a suitable target for disrupting BCL11A-mediated foetal-to-adult globin switching.


Assuntos
Proteínas de Transporte/genética , Globinas/genética , Proteínas Nucleares/genética , Algoritmos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Simulação por Computador , Bases de Dados de Ácidos Nucleicos , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , Globinas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Repressoras , Homologia de Sequência de Aminoácidos , Dedos de Zinco/genética
16.
Metallomics ; 11(5): 906-913, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-30734813

RESUMO

Here, we report the preparation and photo-physical characterization of hexa-coordinated vertebrate globins, human neuroglobin (hNgb) and cytoglobin (hCygb), with the native iron protoporphyrin IX (FePPIX) cofactor replaced by a fluorescent isostructural analogue, zinc protoporphyrin IX (ZnPPIX). To facilitate insertion of ZnPPIX into hexa-coordinated globins, apoproteins prepared via butanone extraction were unfolded by the addition of GuHCl and subsequently slowly refolded in the presence of ZnPPIX. The absorption/emission spectra of ZnPPIX reconstituted hCygb are similar to those observed for ZnPPIX reconstituted myoglobin whereas the absorption and emission spectra of ZnPPIX reconstituted hNgb are blue shifted by ∼2 nm. Different steady state absorption and emission properties of ZnPPIX incorporated in hCygb and hNgb are consistent with distinct hydrogen bonding interactions between ZnPPIX and the globin matrix. The fluorescence lifetime of ZnPPIX in hexa-coordinated globins is bimodal pointing towards increased heterogeneity of the heme binding cavity in hCygb and hNgb. ZnPPIX reconstituted Ngb binds to cytochrome c with the same affinity as reported for the native protein, suggesting that fluorescent analogues of Cygb and Ngb can be readily employed to monitor interactions between vertebrate hexa-coordinated globins and other proteins.


Assuntos
Globinas/química , Heme/análogos & derivados , Protoporfirinas/metabolismo , Vertebrados/metabolismo , Animais , Dicroísmo Circular , Citocromos c/metabolismo , Cavalos , Humanos , Conformação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
17.
Arch Toxicol ; 93(3): 603-613, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30666356

RESUMO

Ethylene oxide (EO), a genotoxic industrial chemical and sterilant, forms covalent adducts with DNA and also with nucleophilic amino acids in proteins. The adduct with N-terminal valine in globin [N-(2-hydroxyethyl)valine (HEV)] has been used in biomonitoring of cumulative exposures to EO. Here we studied in rats the fate of EO-adducted N-termini of globin after life termination of the erythrocytes. Rat erythrocytes were incubated with EO to produce the HEV levels in globin at 0.4-13.2 µmol/g as determined after acidic hydrolysis. Alternative hydrolysis of the isolated globin with enzyme pronase afforded N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) and N-(2-hydroxyethyl)-L-valyl-L-histidine (HEVH), the EO-adducted N-terminal dipeptides of rat globin α- and ß-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the ß-chain. The EO-modified erythrocytes were then given intravenously to the recipient rats. HEVL and HEVH were found to be the ultimate cleavage products excreted in the rat urine. Finally, rats were dosed intraperitoneally with EO, 50 mg/kg. Herein, the initial level of globin-bound HEVL (11.7 ± 1.3 nmol/g) decreased almost linearly over 60 days corresponding to the life span of rat erythrocytes. Daily urinary excretion of HEVL was almost constant for 30-40 days, decreasing faster in the subsequent phase of elimination. Recoveries of the total urinary HEVL from its globin-bound form were 84 ± 6% and 101 ± 17% after administrations of EO and the EO-modified erythrocytes, respectively. In conclusion, urinary HEVL appears to be a promising novel non-invasive biomarker of human exposures to EO.


Assuntos
Óxido de Etileno/toxicidade , Substâncias Perigosas/toxicidade , Animais , Biomarcadores/urina , Monitoramento Ambiental , Eritrócitos , Globinas/metabolismo , Hidrólise , Leucina , Ratos , Valina/química
18.
Genome Biol Evol ; 11(3): 597-612, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668717

RESUMO

Multicellular organisms depend on oxygen-carrying proteins to transport oxygen throughout the body; therefore, proteins such as hemoglobins (Hbs), hemocyanins, and hemerythrins are essential for maintenance of tissues and cellular respiration. Vertebrate Hbs are among the most extensively studied proteins; however, much less is known about invertebrate Hbs. Recent studies of hemocyanins and hemerythrins have demonstrated that they have much wider distributions than previously thought, suggesting that oxygen-binding protein diversity is underestimated across metazoans. Hexagonal bilayer hemoglobin (HBL-Hb), a blood pigment found exclusively in annelids, is a polymer comprised up to 144 extracellular globins and 36 linker chains. To further understand the evolutionary history of this protein complex, we explored the diversity of linkers and extracellular globins from HBL-Hbs using in silico approaches on 319 metazoan and one choanoflagellate transcriptomes. We found 559 extracellular globin and 414 linker genes transcribed in 171 species from ten animal phyla with new records in Echinodermata, Hemichordata, Brachiopoda, Mollusca, Nemertea, Bryozoa, Phoronida, Platyhelminthes, and Priapulida. Contrary to previous suggestions that linkers and extracellular globins emerged in the annelid ancestor, our findings indicate that they have putatively emerged before the protostome-deuterostome split. For the first time, we unveiled the comprehensive evolutionary history of metazoan HBL-Hb components, which consists of multiple episodes of gene gains and losses. Moreover, because our study design surveyed linkers and extracellular globins independently, we were able to cross-validate our results, significantly reducing the rate of false positives. We confirmed that the distribution of HBL-Hb components has until now been underestimated among animals.


Assuntos
Globinas/genética , Invertebrados/genética , Filogenia , Animais
19.
Arch Toxicol ; 93(1): 137-147, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30327826

RESUMO

1,2,4-Benzenetriol (BT) is one of the phenolic metabolites of benzene, a general occupational hazard and ubiquitous environmental air pollutant with leukemogenic potential in humans. Previous studies have revealed that the benzene metabolites phenol and hydroquinone can inhibit hemin-induced erythroid differentiation in K562 cells. We investigated the roles of DNA methylation and histone acetylation in BT-inhibited erythroid differentiation in K562 cells. When K562 cells were treated with 0, 5, 10, 15 or 20 µM BT for 72 h, hemin-induced hemoglobin synthesis decreased in a concentration-dependent manner. Both 5-aza-2'-deoxycytidine (5-aza-CdR, DNA methyltransferase inhibitor) and trichostatin A (TSA, histone deacetylases inhibitor) could prevent 20 µM BT from inhibiting hemin-induced hemoglobin synthesis and the mRNA expression of erythroid genes. Exposure to BT changed DNA methylation levels at several CpG sites of erythroid-specific genes, as well as the acetylation of histone H3 and H4, chromatin occupancy of GATA-1 and recruitment of RNA polymerase II at α-globin and ß-globin gene clusters after hemin induction. These results demonstrated that BT could inhibit hemin-induced erythroid differentiation, where DNA methylation and histone acetylation also played important roles by down-regulating erythroid-specific genes. This partly explained the mechanisms of benzene hematotoxicity.


Assuntos
Benzeno/toxicidade , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA , Histonas/química , Acetilação , Azacitidina/farmacologia , Fator de Transcrição GATA1 , Globinas/genética , Hemina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hidroquinonas , Ácidos Hidroxâmicos/farmacologia , Células K562 , RNA Polimerase II
20.
Microbiologyopen ; 8(2): e00631, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29656507

RESUMO

In the industrial production of xanthan gum using Xanthomonas campestris CGMCC15155, large amounts of ethanol are required to extract xanthan gum from the fermentation broth and remove xanthomonadin impurities. To reduce the amount of ethanol and the overall production cost of xanthan gum, a xanthomonadin-deficient strain of CGMCC15155 was constructed by inserting the Vitreoscilla globin (vgb) gene, under the control of the LacZ promoter, into the region of the pigA gene, which is involved in xanthomonadin synthesis. The insertion of vgb inactivated pigA, resulting in the production of white xanthan gum. The lack of xanthomonadins resulted in a decreased yield of xanthan gum. However, the expression product of vgb gene, VHb, could increase the metabolism of X. campestris, which allowed the production of xanthan gum to reach wild-type levels in the engineered strain. The yield, molecular weight, and rheological properties of the xanthan gum synthesized by the engineered and wild-type bacteria were essentially the same. When the same volume of ethanol was used, the whiteness values of the xanthan gum extracted from engineered and wild-type bacteria were 65.20 and 38.17, respectively. To extract xanthan gum with the same whiteness, three and seven times the fermentation volume of ethanol was required for the engineered and wild-type strains, respectively. Thus, the engineered train reduced the requirement for ethanol in xanthan gum production by 133.3%. The results demonstrated that the engineered bacteria used less ethanol, thus reducing the downstream processing cost in xanthan gum production.


Assuntos
Vias Biossintéticas/genética , Aditivos Alimentares/metabolismo , Engenharia Metabólica , Polissacarídeos Bacterianos/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismo , Biotecnologia/métodos , Aditivos Alimentares/isolamento & purificação , Globinas/genética , Mutagênese Insercional , Polissacarídeos Bacterianos/isolamento & purificação , Vitreoscilla/genética
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