Expressão, purificação e caracterização estrutural inicial do receptor órfão nuclear NOR-1 de rato

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em BiotecnologiaO receptor órfão nuclear NOR-1 é um membro uma superfamília composta por um grupo de fatores de transcrição envolvidos na resposta a esteróides, ácidos graxos, ácidos retinóicos, e outras moléculas lipofílicas. A subfamília de NOR-1está implicada na proliferação celular, diferenciação, apoptose, condrosarcomas, processos inflamatórios e de aterogênese. O receptor NOR-1 é um órfão de ligante atuando sobre a transativação gênica. Até hoje, nenhum ligante é conhecido para este receptor. A estrutura tridimensional do domínio LBD homólogo, Nurr1, foi resolvida através de técnicas de cristalografia. Surpreendentemente, a estrutura não apresentou a cavidade típica de interação com ligantes, nem um sítio de ligação clássico para co-fatores. Com a finalidade de estudar estruturalmente um outro membro da subfamília NR4, dois de seus domínios AF-1 e LBD foram os focos deste trabalho, realizando-se experimentos de expressão, purificação, e análises preliminares de estrutura por UV e Dicroísmo Circular (CD). O domínio LBD de NOR-1 foi purificado com alto grau de pureza, rendendo 3 mg/litro de meio de cultivo e sua estrutura foi avaliada por UV e CD apresentando um conteúdo de a-hélices de 52 % compatível a estrutura 3D do seu homólogo Nurr1. A desnaturação térmica, monitorada por UV e CD, sugere um correto enovelamento para proteína LBD recombinante

Structural stability of Staphylococcus xylosus lipase is modulated by Zn2+ ions

AbstractLipases are well-known enzymes extensively used in industrial biotransformation processes. Besides, their structural and catalytic characteristics have attracted increasing attention of several industries in the last years. In this work, we used biophysical and molecular modeling tools to assess structural properties of Staphylococcus xylosus lipase (SXL). We studied the thermal unfolding of this protein and its zinc-dependent thermotolerance. We demonstrated that SXL is able to be active and stable at moderate temperatures, but this feature is only acquired in the presence of Zn2+. Such characteristic indicates SXL as a zinc-dependent metallolipase

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, β-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.573949/2008-5, 307467/2013-9, 573949/2008-5, 406104/2013-1info:eu-repo/semantics/publishedVersio

Characterization of a fatty acid-binding protein from the Pacific oyster (Crassostrea gigas): pharmaceutical and toxicological implications

Pharmaceuticals and their metabolites constitute a class of xenobiotics commonly found in aquatic environments which may cause toxic effects in aquatic organisms. Several different lipophilic molecules, including some pharmaceuticals, can bind to fatty acid-binding proteins (FABPs), a group of evolutionarily related cytoplasmic proteins that belong to the intracellular lipid-binding protein (iLBP) family. An oyster FABP genome-wide investigation was not available until a recent study on gene organization, protein structure, and phylogeny of Crassostrea gigas iLBPs. Higher transcript levels of the C. gigas FABP2 gene were found after exposure to sewage and pharmaceuticals. Because of its relevance as a potential biomarker of aquatic contamination, in this study, recombinant FABP2 from C. gigas (CgFABP2) was successfully cloned, expressed, and purified, and in vitro and in silico assays were performed using lipids and pharmaceuticals. This is the first characterization of a protein from the iLBP family in C. gigas. Homology modeling and molecular docking were used to evaluate the binding affinities of natural ligands (palmitic, oleic, and arachidonic acids) and pharmaceuticals (ibuprofen, sodium diclofenac, and acetaminophen). Among the tested fatty acids, CgFABP2 showed preference for palmitic acid. The selected pharmaceuticals presented a biphasic-binding mode, suggesting a different binding affinity with a preference for diclofenac. Therefore, the approach using circular dichroism and in silico data might be useful for ligand-binding screening in an invertebrate model organism.info:eu-repo/semantics/publishedVersio

Spectroscopic and catalytic characterization of a functional (FeFeII)-Fe-III biomimetic for the active site of uteroferrin and protein cleavage

A mixed-valence complex, [Fe(III)Fe(II)L1(mu-OAc)(2)]BF4 center dot H2O, where the ligand H(2)L1 = 2-{[[3-[((bis-(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl](pyridin-2-ylmethyl)amino]methyl]phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The (FeFeII)-Fe-III and Fe-2(III) oxidized species were studied spectroelectrochemically. The temperature-dependent population of the S = 3/2 spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (H = -2JS(1).S-2, where J = -5.6 cm(-1)) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The (FeFeII)-Fe-III complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems

Spectroscopic and Catalytic Characterization of a Functional Fe^IIIFe^II Biomimetic for the Active Site of Uteroferrin and Protein Cleavage

A mixed-valence complex, [Fe^IIIFe^II<b>L1</b>(μ-OAc)₂]­BF₄·H₂O, where the ligand H₂<b>L1</b> = 2-{[[3-[((bis­(pyridin-2-ylmethyl)­amino)­methyl)-2-hydroxy-5-methylbenzyl]­(pyridin-2-ylmethyl)­amino]­methyl]­phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The Fe^IIIFe^II and Fe^III₂ oxidized species were studied spectroelectrochemically. The temperature-dependent population of the <i>S</i> = ³/₂ spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (<i>H</i> = −2<i>JS</i>₁·<i>S</i>₂, where <i>J</i> = −5.6 cm^–1) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The Fe^IIIFe^II complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin–lattice relaxation. However, a phosphate-bound Fe^III₂ complex showed an EPR spectrum due to population of the <i>S</i>_tot = 3 state (<i>J</i>= −3.5 cm^–1). The phosphatase activity of the Fe^IIIFe^II complex in hydrolysis of bis­(2,4-dinitrophenyl)­phosphate (<i>k</i>_cat. = 1.88 × 10^–3 s^–1; <i>K</i>_m = 4.63 × 10^–3 mol L^–1) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe^III. It is interesting to note that aqueous solutions of [Fe^IIIFe^II<b>L1</b>(μ-OAc)₂]⁺ are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex’s catalytic promiscuity

Spectroscopic and Catalytic Characterization of a Functional Fe^IIIFe^II Biomimetic for the Active Site of Uteroferrin and Protein Cleavage

A mixed-valence complex, [Fe^IIIFe^II<b>L1</b>(μ-OAc)₂]­BF₄·H₂O, where the ligand H₂<b>L1</b> = 2-{[[3-[((bis­(pyridin-2-ylmethyl)­amino)­methyl)-2-hydroxy-5-methylbenzyl]­(pyridin-2-ylmethyl)­amino]­methyl]­phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The Fe^IIIFe^II and Fe^III₂ oxidized species were studied spectroelectrochemically. The temperature-dependent population of the <i>S</i> = ³/₂ spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (<i>H</i> = −2<i>JS</i>₁·<i>S</i>₂, where <i>J</i> = −5.6 cm^–1) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The Fe^IIIFe^II complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin–lattice relaxation. However, a phosphate-bound Fe^III₂ complex showed an EPR spectrum due to population of the <i>S</i>_tot = 3 state (<i>J</i>= −3.5 cm^–1). The phosphatase activity of the Fe^IIIFe^II complex in hydrolysis of bis­(2,4-dinitrophenyl)­phosphate (<i>k</i>_cat. = 1.88 × 10^–3 s^–1; <i>K</i>_m = 4.63 × 10^–3 mol L^–1) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe^III. It is interesting to note that aqueous solutions of [Fe^IIIFe^II<b>L1</b>(μ-OAc)₂]⁺ are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex’s catalytic promiscuity