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1.
Molecules ; 26(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562280

RESUMO

Oxidative protein folding is a biological process to obtain a native conformation of a protein through disulfide-bond formation between cysteine residues. In a cell, disulfide-catalysts such as protein disulfide isomerase promote the oxidative protein folding. Inspired by the active sites of the disulfide-catalysts, synthetic redox-active thiol compounds have been developed, which have shown significant promotion of the folding processes. In our previous study, coupling effects of a thiol group and guanidyl unit on the folding promotion were reported. Herein, we investigated the influences of a spacer between the thiol group and guanidyl unit. A conjugate between thiol and guanidyl units with a diethylene glycol spacer (GdnDEG-SH) showed lower folding promotion effect compared to the thiol-guanidyl conjugate without the spacer (GdnSH). Lower acidity and a more reductive property of the thiol group of GdnDEG-SH compared to those of GdnSH likely resulted in the reduced efficiency of the folding promotion. Thus, the spacer between the thiol and guanidyl groups is critical for the promotion of oxidative protein folding.


Assuntos
Etilenoglicol/química , Estresse Oxidativo/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/química , Compostos de Sulfidrila/química , Catálise , Cisteína/química , Dissulfetos/química , Etilenoglicol/farmacologia , Glutationa/química , Cinética , Oxirredução/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Compostos de Sulfidrila/farmacologia
2.
Biochem Biophys Res Commun ; 545: 119-124, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33548624

RESUMO

Aß42 aggregation plays a central role in the pathogenesis of Alzheimer's disease. In addition to the insoluble fibrils that comprise the amyloid plaques, Aß42 also forms soluble aggregates collectively called oligomers, which are more toxic and pathogenic than fibrils. Understanding the structure and dynamics of Aß42 oligomers is critical for developing effective therapeutic interventions against these oligomers. Here we studied the structural dynamics of Aß42 globulomers, a type of Aß42 oligomers prepared in the presence of sodium dodecyl sulfate, using site-directed spin labeling. Spin labels were introduced, one at a time, at all 42 residue positions of Aß42 sequence. Electron paramagnetic resonance spectra of spin-labeled samples reveal four structural segments based on site-dependent spin label mobility pattern. Segment-1 consists of residues 1-6, which have the highest mobility that is consistent with complete disorder. Segment-3 is the most immobilized region, including residues 31-34. Segment-2 and -4 have intermediate mobility and are composed of residues 7-30 and 35-42, respectively. Considering the inverse relationship between protein dynamics and stability, our results suggest that residues 31-34 are the most stable segment in Aß42 oligomers. At the same time, the EPR spectral lineshape suggests that Aß42 globulomers lack a well-packed structural core akin to that of globular proteins.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Marcadores de Spin
3.
Nat Commun ; 12(1): 807, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547325

RESUMO

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.


Assuntos
Apoproteínas/química , Cálcio/química , Calmodulina/química , Hipertermia Maligna/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Substituição de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Cisteína/metabolismo , Expressão Gênica , Humanos , Transporte de Íons , Hipertermia Maligna/genética , Hipertermia Maligna/patologia , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Suínos
4.
Nat Commun ; 12(1): 581, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495458

RESUMO

Formaldehyde (FA) has long been considered as a toxin and carcinogen due to its damaging effects to biological macromolecules, but its beneficial roles have been increasingly appreciated lately. Real-time monitoring of this reactive molecule in living systems is highly desired in order to decipher its physiological and/or pathological functions, but a genetically encoded FA sensor is currently lacking. We herein adopt a structure-based study of the underlying mechanism of the FA-responsive transcription factor HxlR from Bacillus subtilis, which shows that HxlR recognizes FA through an intra-helical cysteine-lysine crosslinking reaction at its N-terminal helix α1, leading to conformational change and transcriptional activation. By leveraging this FA-induced intra-helical crosslinking and gain-of-function reorganization, we develop the genetically encoded, reaction-based FA sensor-FAsor, allowing spatial-temporal visualization of FA in mammalian cells and mouse brain tissues.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Formaldeído/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/química , Encéfalo/metabolismo , Reagentes para Ligações Cruzadas/química , Cisteína/química , Cisteína/metabolismo , Formaldeído/análise , Humanos , Lisina/química , Lisina/metabolismo , Camundongos , Conformação Proteica , Reprodutibilidade dos Testes , Fatores de Transcrição/química
5.
Arch Biochem Biophys ; 699: 108748, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444627

RESUMO

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.


Assuntos
Apolipoproteína A-I/química , Corantes Fluorescentes/química , Sondas Moleculares/química , Multimerização Proteica , Pirenos/química , Apolipoproteína A-I/genética , Cisteína/química , Humanos , Mutação , Espectrometria de Fluorescência
6.
Biochem Biophys Res Commun ; 540: 101-107, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33460839

RESUMO

Pyrrolidone carboxypeptidases (Pcps) (E.C. 3.4.19.3) can cleave the peptide bond adjacent to pyro-glutamic acid (pGlu), an N-terminal modification observed in some proteins that provides protection against common proteases. Pcp derived from extremely thermophilic Fervidobacterium islandicum AW-1 (FiPcp), that belongs to the cysteine protease family, is involved in keratin utilization under stress conditions. Although an irreversible oxidative modification of active cysteine to its sulfonic acid derivative (Cys-SO3H) renders the enzyme inactive, the molecular details for the sulfonic acid modification in inactive Pcp remain unclear. Here, we determined the crystal structure of FiPcp at 1.85 Å, revealing the oxidized form of cysteine sulfonic acid (C156-SO3H) in the catalytic triad (His-Cys-Glu), which participates in the hydrolysis of pGlu residue containing peptide bond. The three oxygen atoms of cysteine sulfonic acid were stabilized by hydrogen bonds with H180, carbonyl backbone of Q83, and water molecules, resulting in inactivation of FiPcp. Furthermore, FiPcp demonstrated a unique 139KKKK142 motif involved in inter-subunit electrostatic interactions whose mutation significantly affects the thermostability of tetrameric FiPcp. Thus, our high-resolution structure of the first inactive FiPcp with irreversible oxidative modification of active cysteine provides not only the molecular basis of the redox-dependent catalysis of Pcp, but also the structural features of its thermostability.


Assuntos
Bactérias/enzimologia , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Queratinas/metabolismo , Pirrolidinonas/metabolismo , Motivos de Aminoácidos , Bactérias/classificação , Domínio Catalítico , Cristalografia por Raios X , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Estabilidade Enzimática , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Eletricidade Estática , Água/metabolismo
7.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440688

RESUMO

Hydrophobins are a family of small secreted proteins found exclusively in fungi, and they play various roles in the life cycle. In the present study, genome wide analysis and transcript profiling of the hydrophobin family in Cordyceps militaris, a well-known edible and medicinal mushroom, were studied. The distribution of hydrophobins in ascomycetes with different lifestyles showed that pathogenic fungi had significantly more hydrophobins than saprotrophic fungi, and class II members accounted for the majority. Phylogenetic analysis of hydrophobin proteins from the species of Cordyceps s.l. indicated that there was more variability among the class II members than class I. Only a few hydrophobin-encoding genes evolved by duplication in Cordyceps s.l., which was inconsistent with the important role of gene duplication in basidiomycetes. Different transcript patterns of four hydrophobin-encoding genes during the life cycle indicated the possible different functions for each. The transcripts of Cmhyd2, 3 and 4 can respond to light and were related with the photoreceptors. CmQHYD, with four hydrophobin II domains, was first found in C. militaris, and multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae. These results could be helpful for further function research of hydrophobins and could provide valuable information for the evolution of hydrophobins.


Assuntos
Cordyceps/classificação , Cordyceps/genética , Cisteína/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Genômica , Sequência de Aminoácidos , Cordyceps/crescimento & desenvolvimento , Cisteína/química , Carpóforos/genética , Proteínas Fúngicas/química , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica/métodos , Luz , Filogenia , Domínios Proteicos , Transcriptoma
8.
Molecules ; 26(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466352

RESUMO

Based on a set of six vector properties, the partial correlation diagram is calculated for a set of 28 S-alkylcysteine diazomethyl- and chloromethyl-ketone derivatives. Those with the greatest antileukemic activity in the same class correspond to high partial correlations. A periodic classification is performed based on information entropy. The first four characteristics denote the group, and the last two indicate the period. Compounds in the same period and, especially, group present similar properties. The most active substances are situated at the bottom right. Nine classes are distinguished. The principal component analysis of the homologous compounds shows five subclasses included in the periodic classification. Linear fits of both antileukemic activities and stability are good. They are in agreement with the principal component analysis. The variables that appear in the models are those that show positive loading in the principal component analysis. The most important properties to explain the antileukemic activities (50% inhibitory concentration Molt-3 T-lineage acute lymphoblastic leukemia minus the logarithm of 50% inhibitory concentration Nalm-6 B-lineage acute lymphoblastic leukemia and stability k) are ACD logD, surface tension and number of violations of Lipinski's rule of five. After leave-m-out cross-validation, the most predictive model for cysteine diazomethyl- and chloromethyl-ketone derivatives is provided.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Cisteína/química , Cetonas/química , Cetonas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proliferação de Células , Entropia , Humanos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Relação Quantitativa Estrutura-Atividade , Células Tumorais Cultivadas
9.
Methods Mol Biol ; 2186: 51-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32918729

RESUMO

Single-channel planar lipid bilayer (PLB) recording of bacterial porins has revealed molecular details of transport across the outer membrane of Gram-negative bacteria, including antibiotic permeation and protein translocation. To explore directional transport processes across cellular membranes, the orientation of porins or other pore-forming proteins must be established in a lipid bilayer prior to experimentation. Here, we describe a direct method for determining the orientation of porins in a PLB-with a focus on E. coli OmpF-by using targeted covalent modification of cysteine mutants. Each of the two possible orientations can be correlated with the porin conductance asymmetry, such that thereafter an I-V curve taken at the start of an experiment will suffice to establish orientation.


Assuntos
Eletrofisiologia/métodos , Escherichia coli/metabolismo , Bicamadas Lipídicas/química , Porinas/química , Porinas/fisiologia , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana , Mutação , Porinas/genética , Porinas/metabolismo , Transporte Proteico
10.
Biochem Biophys Res Commun ; 534: 27-33, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310184

RESUMO

Protein labeling with a functional molecule is a technique widely used for protein research. The covalent reaction of self-labeling peptide tags with synthetic probe-modified small molecules enables tag-fused protein labeling with chemically diverse molecules, including fluorescent probes. We report the discovery, by in vitro directed evolution, of a novel 23-mer dibenzocyclooctyne (DBCO)-reactive peptide (DRP) tag using Systematic Evolution of Ligands by EXponential enrichment (SELEX) with a combination of a reconstituted cell-free translation system (PURE system) and cDNA display. The N- and C-terminal DRP truncations created a shorter 16-mer DBCO-reactive peptide (sDRP) tag without significant reactivity reduction. By fusing the sDRP tag to a model protein, we showed the chemical labeling and in-gel fluorescence imaging of the sDRP-fused protein using a fluorescent DBCO probe. Results showed that sDRP tag-mediated protein labeling has potential for use as a basic molecular tool in a variety of applications for protein research.


Assuntos
Evolução Molecular Direcionada/métodos , Peptídeos/química , Ciclo-Octanos/química , Ciclo-Octanos/metabolismo , Cisteína/química , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Imagem Molecular/métodos , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química
11.
Food Chem ; 338: 127787, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32827901

RESUMO

A rapid colorimetric method using cysteine-modified silver nanoparticles (Cys-AgNPs) is applied for the detection of 3-monochloropropane-1,2-diol (3-MCPD). Indeed, in the presence of 3-MCPD, the color of Cys-AgNPs solution changes from yellow to pink within five minutes at 100 °C and pH 9.3. This change is mainly attributed to the ability of amino group of cysteine to react with 3-MCPD to form N-(2,3-dihydroxypropyl)-amino acid grafted on AgNPs (3-MCPD-Cys-AgNPs) in alkaline medium. This color change makes 3-MCPD to be clearly detectable by unassisted visual means even at 0.1 µg⋅mL-1. Besides, using UV-Vis spectroscopic technique, a linear range from 0.1 µg⋅mL-1 to 1.25 µg⋅mL-1 for 3-MCPD detection is obtained, with a calculated detection limit of 0.084 µg⋅mL-1. These results suggest that this sensing technique is sensitive to 3-MCPD and may have a substantial application in the rapid detection of food contaminants particularly, where quality and safety of food products are paramount concern.


Assuntos
Cisteína/química , Análise de Alimentos/métodos , Nanopartículas Metálicas/química , Prata/química , alfa-Cloridrina/análise , Colorimetria , Contaminação de Alimentos/análise , Fatores de Tempo , alfa-Cloridrina/isolamento & purificação
12.
Food Chem ; 334: 127611, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32712493

RESUMO

Plant polyphenols applied as natural antioxidant ingredients, are known to bind to cysteine residues on meat proteins. The aim of this study was to examine the effect of light exposure on the formation of cysteine-phenol adduct in meat added 4-methylcatechol (4MC), a model polyphenol, during storage through quantitative LC-MS/MS-based analysis. Cysteine-4-methylcatechol adduct (Cys-4MC) formation in meat added 1500 ppm 4-MC increased significantly (by 50%) when stored under light in oxygen at 4 °C for 7 days as compared to storage in the dark. This was reflected by a significant decrease in thiol concentrations in the same sample. Gel electrophoresis showed loss in myosin heavy chain (MHC), and a resulting increase in cross-linked MHC (CL-MHC) and larger protein polymers in samples added 4MC. Protein blots stained with nitroblue tetrazolium (NBT) showed intensive protein-polyphenol binding in the meat samples added 4MC, but no major differences between storage conditions.


Assuntos
Catecóis/química , Armazenamento de Alimentos/métodos , Proteínas de Carne/química , Carne , Oxigênio/química , Antioxidantes/química , Cromatografia Líquida , Cisteína/química , Eletroforese em Gel de Poliacrilamida , Luz , Carne/análise , Proteínas de Carne/metabolismo , Cadeias Pesadas de Miosina/química , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem
13.
Food Chem ; 345: 128761, 2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-33310557

RESUMO

2-Acetylthiazole possesses a nutty, cereal-like and popcorn-like aroma and a low odor threshold, and this compound has been identified in some processed foods, while the formation pathway of 2-acetylthiazole has not been clearly elucidated. Here, a model reaction of d-glucose and l-cysteine was constructed to investigate the formation pathway of 2-acetylthiazole. l-Cysteine, d-glucose and the corresponding intermediates, namely, dicarbonyl compounds (DCs), were involved in the formation of 2-acetylthiazole and detected by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS), high-performance ion chromatography (HPIC) and HPLC, respectively. The carbon module labeling (CAMOLA) technique revealed that the C-4 and C-5 of 2-acetylthiazole were derived from the carbons of glucose. The potential of glyoxal, which is degraded by glucose, to form 2-acetylthiazole was revealed for the first time. A novel route to form 2-acetylthiazole by the reaction of glyoxal and methylglyoxal produced by d-glucose with H2S and NH3 produced by l-cysteine was proposed.


Assuntos
Reação de Maillard , Tiazóis/síntese química , Cromatografia Líquida de Alta Pressão , Cisteína/química , Glucose/química , Glioxal/química , Odorantes/análise , Aldeído Pirúvico/química , Espectrometria de Massas em Tandem
14.
Food Chem ; 338: 127928, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32919374

RESUMO

We investigated the inhibitory effect and binding mechanism of four selected compounds (ascorbic acid, l-cysteine, glutathione, and citric acid) on membrane-bound polyphenol oxidases (mPPO) using spectroscopic and molecular docking techniques. Kinetic analysis demonstrated that these inhibitors reversibly inhibited the mPPO activity. Fluorescence spectroscopy revealed that the intrinsic fluorescence intensity of mPPO was quenched by inhibitors with a single class of the inhibition site on mPPO. Amino acid residues His 180, His 201, His 366, Cys 184, Glu 328, and Asn 333 were the important binding sites in the active center. These sites were identified using molecular docking techniques. Our findings suggested that the inhibitors were allosterically bound to the active center of mPPO through hydrogen bonds and ion contacts. This study provides new insights into the active site residues responsible for catalyzing mPPO and provides applicable information about the design of mPPO inhibitors.


Assuntos
Catecol Oxidase/metabolismo , Malus/enzimologia , Simulação de Acoplamento Molecular , Proteínas de Plantas/metabolismo , Regulação Alostérica , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Sítios de Ligação , Catecol Oxidase/antagonistas & inibidores , Cisteína/química , Cisteína/metabolismo , Glutationa/química , Glutationa/metabolismo , Cinética , Proteínas de Plantas/antagonistas & inibidores , Espectrometria de Fluorescência
15.
Biochemistry ; 59(51): 4793-4798, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33326741

RESUMO

Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.


Assuntos
Proteínas de Bactérias/química , Cisteína/análogos & derivados , Flavina-Adenina Dinucleotídeo/química , Glucosamina/análogos & derivados , NADP/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Bacillus cereus/enzimologia , Cristalografia por Raios X , Cisteína/química , Glucosamina/química , Oxirredução , Estrutura Quaternária de Proteína , Staphylococcus aureus/enzimologia
16.
J Am Chem Soc ; 142(52): 21883-21890, 2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33320670

RESUMO

The SARS coronavirus 2 (SARS-CoV-2) main protease (Mpro) is an attractive broad-spectrum antiviral drug target. Despite the enormous progress in structure elucidation, the Mpro's structure-function relationship remains poorly understood. Recently, a peptidomimetic inhibitor has entered clinical trial; however, small-molecule orally available antiviral drugs have yet to be developed. Intrigued by a long-standing controversy regarding the existence of an inactive state, we explored the proton-coupled dynamics of the Mpros of SARS-CoV-2 and the closely related SARS-CoV using a newly developed continuous constant pH molecular dynamics (MD) method and microsecond fixed-charge all-atom MD simulations. Our data supports a general base mechanism for Mpro's proteolytic function. The simulations revealed that protonation of His172 alters a conserved interaction network that upholds the oxyanion loop, leading to a partial collapse of the conserved S1 pocket, consistent with the first and controversial crystal structure of SARS-CoV Mpro determined at pH 6. Interestingly, a natural flavonoid binds SARS-CoV-2 Mpro in the close proximity to a conserved cysteine (Cys44), which is hyper-reactive according to the CpHMD titration. This finding offers an exciting new opportunity for small-molecule targeted covalent inhibitor design. Our work represents a first step toward the mechanistic understanding of the proton-coupled structure-dynamics-function relationship of CoV Mpros; the proposed strategy of designing small-molecule covalent inhibitors may help accelerate the development of orally available broad-spectrum antiviral drugs to stop the current pandemic and prevent future outbreaks.


Assuntos
Antivirais/química , Antivirais/farmacologia , /efeitos dos fármacos , /enzimologia , Sítios de Ligação , Cisteína/química , Desenho de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Conformação Proteica , Prótons , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
17.
Nat Commun ; 11(1): 5031, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024112

RESUMO

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.


Assuntos
Evolução Molecular , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Zinco/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Proteínas de Transporte Vesicular/genética , Dedos de Zinco
18.
Biol Direct ; 15(1): 19, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33066821

RESUMO

The spike glycoprotein of the SARS-CoV-2 virus, which causes COVID-19, has attracted attention for its vaccine potential and binding capacity to host cell surface receptors. Much of this research focus has centered on the ectodomain of the spike protein. The ectodomain is anchored to a transmembrane region, followed by a cytoplasmic tail. Here we report a distant sequence similarity between the cysteine-rich cytoplasmic tail of the coronavirus spike protein and the hepcidin protein that is found in humans and other vertebrates. Hepcidin is thought to be the key regulator of iron metabolism in humans through its inhibition of the iron-exporting protein ferroportin. An implication of this preliminary observation is to suggest a potential route of investigation in the coronavirus research field making use of an already-established literature on the interplay of local and systemic iron regulation, cytokine-mediated inflammatory processes, respiratory infections and the hepcidin protein. The question of possible homology and an evolutionary connection between the viral spike protein and hepcidin is not assessed in this report, but some scenarios for its study are discussed.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Hepcidinas/genética , Ferro/metabolismo , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/genética , Animais , Proteínas de Transporte de Cátions/metabolismo , Cisteína/química , Citocinas/metabolismo , Citoplasma/metabolismo , Hepcidinas/química , Humanos , Hipóxia , Inflamação , Interleucina-6/metabolismo , Pandemias , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Tetraodontiformes
19.
Environ Sci Process Impacts ; 22(9): 1852-1864, 2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32966465

RESUMO

Carbonyl sulfide (COS) and carbon disulfide (CS2) are important atmospheric gases that are formed from organic sulfur precursors present in natural waters when exposed to sunlight. However, it remains unclear how specific water constituents, such as dissolved organic matter (DOM), affect COS and CS2 formation. To better understand the role of DOM, irradiation experiments were conducted in O2-free synthetic waters containing four different DOM isolates, acquired from freshwater to open ocean sources, and the sulfur-based amino acid, cysteine (CYS). CYS is a known natural precursor of COS and CS2. Results indicated that COS formation did not vary strongly with DOM type, although small impacts were observed on the kinetic patterns. COS formation also increased with increasing CYS concentration but decreased with increasing DOM concentration. Quenching experiments indicated that ˙OH was not involved in the rate-limiting step of COS formation, whereas excited triplet states of DOM (3CDOM*) were plausibly involved, although the quenching agents used to remove 3CDOM* may have reacted with the CYS-derived intermediates as well. CS2 was not formed under any of the experimental conditions. Overall, DOM-containing synthetic waters had a limited to no effect towards forming COS and CS2, especially when compared to the higher concentrations formed in sunlit natural waters, as examined previously. The reasons behind this limited effect need to be explored further but may be due to the additional water quality constituents present in these natural waters. The findings of this study imply that multiple variables beyond DOM govern COS and CS2 photoproduction when moving from freshwaters to open ocean waters.


Assuntos
Dissulfeto de Carbono , Cisteína/química , Poluentes Químicos da Água/química , Fotólise , Óxidos de Enxofre , Luz Solar , Poluentes Químicos da Água/análise
20.
PLoS One ; 15(9): e0239464, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946534

RESUMO

Omeprazole is the most commonly used proton pump inhibitor (PPI), a class of medications whose therapeutic mechanism of action involves formation of a disulfide linkage to cysteine residues in the H+/K+ ATPase pump on gastric secretory cells. Covalent linkage between the sole sulfur group of omeprazole and selected cysteine residues of the pump protein results in inhibition of acid secretion in the stomach, an effect that ameliorates gastroesophageal reflux and peptic ulcer disease. PPIs, though useful for specific conditions when used transiently, are associated with diverse untoward effects when used long term. The mechanisms underlying these potential off-target effects remain unclear. PPIs may, in fact, interact with non-canonical target proteins (non-pump molecules) resulting in unexpected pathophysiological effects, but few studies describe off-target PPI binding. Here, we describe successful cloning of monoclonal antibodies against protein-bound omeprazole. We developed and used monoclonal antibodies to characterize the protein target range of omeprazole, stability of omeprazole-bound proteins, and the involvement of cysteines in binding of omeprazole to targets. We demonstrate that a wide range of diverse proteins are targeted by omeprazole. Protein complexes, detected by Western blotting, are resistant to heat, detergents, and reducing agents. Reaction of omeprazole occurs with cysteine-free proteins, is not fully inhibited by cysteine alkylation, occurs at neutral pH, and induces protein multimerization. At least two other clinically used PPIs, rabeprazole and tenatoprazole, are capable of binding to proteins in a similar fashion. We conclude that omeprazole binds to multiple proteins and is capable of forming highly stable complexes that are not dependent on disulfide linkages between the drug and protein targets. Further studies made possible by these antibodies may shed light on whether PPI-protein complexes underlie off-target untoward effects of chronic PPI use.


Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Omeprazol/imunologia , Omeprazol/metabolismo , Inibidores da Bomba de Prótons/imunologia , Inibidores da Bomba de Prótons/metabolismo , Animais , Sítios de Ligação , Cisteína/química , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Omeprazol/química , Ligação Proteica , Inibidores da Bomba de Prótons/química
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