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1.
Molecules ; 26(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673598

RESUMO

Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.


Assuntos
Fármacos Antiobesidade/química , Obesidade/tratamento farmacológico , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Tecido Adiposo/efeitos dos fármacos , Alquinos/química , Aminobutiratos/química , Animais , Fármacos Antiobesidade/farmacologia , Apetite/efeitos dos fármacos , Ácido Aspártico/química , Modelos Animais de Doenças , Descoberta de Drogas , Ácido Glutâmico/química , Glicina/análogos & derivados , Glicina/química , Células HEK293 , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Insulina/metabolismo , Lactamas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Relação Estrutura-Atividade , Distribuição Tecidual , Triazóis/química
2.
Plant Mol Biol ; 105(4-5): 451-462, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33387174

RESUMO

KEY MESSAGE: The relative position of domains is critical for enzymatic properties of tau class glutathione S-transferases, and altering the position of linker far away from the active center affects catalytic property. Glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes whose main function is to improve plant resistance to stresses. To understand the structural effects of tau class GSTs on their function, using OsGSTU17 as an example, we predicted the residues involved in the interactions between its domains and linker region. We further detected the structural changes in mutants and the corresponding changes in terms of substrate activity and kinetic parameters. Four pairs of residues, including Ala14 and Trp165, Arg20 and Tyr154, Glu74 and Arg98, Asp77 and Met87, forming hydrogen bonds and salt bridges were found to play important roles in maintaining the relative position between the domains and linker region inside the protein. The hydrogen bond between Trp165 and Ala14 affected the structural stability has been demonstrated in our previous study. The mutant R20A lost almost all catalytic activity. Interestingly, the mutant E74A exhibited a significant decrease in activity towards 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole, 1-chloro-2, 4-dinitrobenzene and 4-nitrobenzyl chloride, while its activity towards substrate cumene hydroperoxide remained unchanged. Compared with other mutants, the mutant D77A exhibited decreased affinity to its substrates and increased activity towards 1-chloro-2, 4-dinitrobenzene and cumene hydroperoxide, but its thermodynamic stability did not change significantly. The relative position of individual domain was critical for enzymatic properties, and the linker which is far away from the active site could change the enzymatic properties of GSTs via altering the relative position of the individual domain. Our results provide insights into the relationship between structure and function of tau class GSTs.


Assuntos
Aminoácidos/genética , Domínio Catalítico , Glutationa Transferase/genética , Oryza/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Arginina/química , Arginina/genética , Arginina/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Estabilidade Enzimática/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Modelos Moleculares , Mutação , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
3.
Life Sci ; 269: 119026, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444617

RESUMO

Morphine is a commonly used opioid drug to treat acute pain by binding to the mu-opioid receptor (MOR), but its effective analgesic efficacy via triggering of the heterotrimeric Gi protein pathway is accompanied by a series of adverse side effects via triggering of the ß-arrestin pathway. Recently, PZM21, a recently developed MOR biased agonist, shows preferentially activating the G protein pathway over ß-arrestin pathway. However, there is no high-resolution receptor structure in complex with PZM21 and its action mechanism remains elusive. In this study, PZM21 and Morphine were docked to the active human MOR-1 homology structure and then subjected to the molecular dynamics (MD) simulations in two different situations (i.e., one situation includes the crystal waters but another does not). Detailed comparisons between the two systems were made to characterize the differences in protein-ligand interactions, protein secondary and tertiary structures and dynamics networks. PZM21 could strongly interact with Y3287.43 of TM7, besides the residues (Asp1493.32 and Tyr1503.33) of TM3. The two systems' network paths to the intracellular end of TM6 were roughly similar but the paths to the end of TM7 were different. The PZM21-bound MOR's intracellular ends of TM5-7 bent outward more along with the distance changes of the three key molecular switches (ionic lock, transmission and Tyr toggle) and the distance increase of some conserved inter-helical residue pairs. The larger intracellular opening of the receptor could potentially facilitate G protein binding.


Assuntos
Simulação de Dinâmica Molecular , Receptores Opioides mu/agonistas , Tiofenos/farmacologia , Ureia/análogos & derivados , Regulação Alostérica , Animais , Ácido Aspártico/química , Análise por Conglomerados , Sequência Conservada , Cristalização , Humanos , Ligantes , Camundongos , Morfina/farmacologia , Análise de Componente Principal , Conformação Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Tiofenos/química , Tirosina/química , Ureia/química , Ureia/farmacologia , Água/química
4.
Int J Nanomedicine ; 15: 8465-8478, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33149587

RESUMO

Introduction: Decellularized matrix from porcine small intestinal submucosa (SIS) endows scaffolds with an ECM-like surface, which enhances stem cell self-renewal, proliferation, and differentiation. Mesoporous bioactive glass (MBG) is extensively recognized as an excellent bio-ceramic for fabricating bone grafts. Materials and Methods: In the current study, SIS was doped on an MBG scaffold (MBG/SIS) using polyurethane foam templating and polydopamine chemistry method. To mimic the bony environment of a natural bone matrix, an ECM-inspired delivery system was constructed by coupling the BMP2-related peptide P28 to a heparinized MBG/SIS scaffold (MBG/SIS-H-P28). The release of P28 from MBG/SIS-H-P28 and its effects on the proliferation, viability, and osteogenic differentiation of bone marrow stromal stem cells were investigated in vitro and in vivo. Results: Our research indicated that the novel tissue-derived ECM scaffold MBG/SIS has a hierarchical and interconnected porous architecture, and superior biomechanical properties. MBG/SIS-H-P28 released P28 in a controlled manner, with the long-term release time of 40 d. The results of in vitro experiments showed improvements in cell proliferation, cell viability, alkaline phosphatase activity, and mRNA expression levels of osteogenesis-related genes (Runx-2, OCN, OPN, and ALP) compared to those of MBG/SIS or MBG/SIS-P28 and MBG/SIS-H-P28. The in vivo results demonstrated that MBG/SIS-H-P28 scaffolds evidently increased bone formation in rat calvarial critical-sized defect compared to that in controls. Conclusion: MBG/SIS-H-P28 scaffolds show potential as ideal platforms for delivery of P28 and for providing a bony environment for bone regeneration.


Assuntos
Ácido Aspártico/química , Materiais Biocompatíveis/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Osso e Ossos/efeitos dos fármacos , Cerâmica/farmacologia , Matriz Extracelular/metabolismo , Osteoblastos/efeitos dos fármacos , Peptídeos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Porosidade , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Suínos , Tecidos Suporte/química
5.
Proc Natl Acad Sci U S A ; 117(41): 25517-25522, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32973095

RESUMO

Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.


Assuntos
Proteínas de Escherichia coli , Prótons , Trocadores de Sódio-Hidrogênio , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Lisina/química , Lisina/genética , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Eletricidade Estática
6.
Chemosphere ; 260: 127600, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32758769

RESUMO

Granules initiation and development is the backbone of aerobic granular sludge technology. Feed composition can notably affect initiation and development of aerobic granules, and yield aerobic granules with distinct microbial community, morphology and structure. This paper reports an unexpected formation of aerobic granules in an aspartic acid fed SBR under unfavorable hydrodynamic selection conditions. Detailed characteristics of these aerobic granules were investigated in terms of morphology, structure, bioactivity and EPS. The results showed that due to the absence of favorable hydrodynamic selection pressure, the formed aerobic granules had an irregular shape with a rough outline and loose internal structure, which was quite different from mature aerobic granules. Bacteria in these aerobic granules were mainly presented in the form of microcolony with calcium and ß-polysaccharides responsible for its mechanical stability. The high N/C ratio of aspartic acid enabled the enrichment of significant amount of nitrifiers within aerobic granules and thus resulted in high nitrification activity of these aerobic granules. The negatively charged and hydrophilic aspartic acid also induced the bacteria to secrete more exopolysaccharides for contributing to more neutral and hydrophilic surface of the aerobic granules, which was beneficial for aspartic acid capture. As a result, polysaccharides, rather than proteins, became the major components of EPS in these aerobic granules. This paper provides us a foundation to better understand the granulation potential of proteinaceous substrates that is frequently encountered in industrial wastewaters.


Assuntos
Ácido Aspártico/química , Reatores Biológicos/microbiologia , Esgotos/microbiologia , Purificação da Água/métodos , Aerobiose , Análise da Demanda Biológica de Oxigênio , China , Matriz Extracelular de Substâncias Poliméricas/química , Hidrodinâmica , Microbiota , Modelos Teóricos , Nitrificação , Proteobactérias/isolamento & purificação , Esgotos/química , Propriedades de Superfície , Águas Residuárias/química
7.
Nat Chem Biol ; 16(9): 1013-1018, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601484

RESUMO

D-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of L-amino acids into their D-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a D-aspartate (D-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/ß-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of L-Asp to D-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH-substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.


Assuntos
Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Família Multigênica , Peptídeos Cíclicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Racemases e Epimerases/genética , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
8.
Proc Natl Acad Sci U S A ; 117(26): 14948-14957, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541034

RESUMO

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3ß-O-l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3ß-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of "higher" fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus We show that a bifunctional enzyme, ergosteryl-3ß-O-l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase-that produces aspartyl-tRNAAsp (Asp-tRNAAsp)-and of a Domain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNAAsp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across "higher" fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.


Assuntos
Ácido Aspártico/metabolismo , Aspergillus fumigatus/metabolismo , RNA Fúngico/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Esteróis/metabolismo , Aminoacilação , Ácido Aspártico/química , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , RNA Fúngico/química , RNA Fúngico/genética , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Esteróis/química
9.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140459, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32474105

RESUMO

In the biological proteins, aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. Asp-residue isomerization causes the aggregation and the insolubilization of proteins, and is considered to be involved in various age-related diseases. Although Suc intermediate was considered to be formed by nucleophilic attack of the main-chain amide nitrogen of N-terminal side adjacent residue to the side-chain carboxyl carbon of Asp residue, previous studies have shown that the nucleophilic attack is more likely to proceed via iminol tautomer when the water molecules act as catalysts. However, the full pathway to Suc-intermediate formation has not been investigated, and the experimental analyses for the Asp-residue isomerization mechanism at atomic and molecular levels, such as the analysis of the transition state geometry, are difficult. In the present study, we computationally explored the full pathways for Suc-intermediate formation from Asp residues. The calculations were performed two types of reactant complexes, and all energy minima and TS geometries were optimized using B3LYP density functional methods. As a result, the SI-intermediate formation was divided into three processes, i.e., iminolization, cyclization, and dehydration processes, and the activation energies were calculated to be 26.1 or 28.4 kcal mol-1. These values reproduce the experimental data. The computational results show that abundant water molecules in living organisms are effective catalysts for the Asp-residue isomerization.


Assuntos
Ácido Aspártico/química , Modelos Químicos , Succinimidas/síntese química , Água/química , Amidas , Catálise , Ciclização , Isomerismo , Modelos Moleculares , Nitrogênio , Proteínas/química
10.
J Med Chem ; 63(9): 4528-4554, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302123

RESUMO

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against human nNOS, and only modest selectivity for nNOS over related enzymes. In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and assayed them against rat and human nNOS, human eNOS, and murine and (in some cases) human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this residue.


Assuntos
Aminoquinolinas/farmacologia , Ácido Aspártico/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Aminoquinolinas/síntese química , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacocinética , Animais , Barreira Hematoencefálica/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Permeabilidade , Ligação Proteica , Ratos , Relação Estrutura-Atividade
11.
J Chromatogr A ; 1623: 461134, 2020 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-32345439

RESUMO

The isomerization of amino acids in peptides and proteins induces structural changes and aggregation. The isomerization rate of aspartic acid (Asp) is high and causes various serious diseases including Alzheimer's disease and cataract. Herein, a method for the comprehensive separation and sensitive detection of isomerized crystallin containing Asp (l-α-Asp, l-ß-Asp, d-α-Asp, and d-ß-Asp) was developed using chiral derivatization and reversed-phase UHPLC separation. Of three candidate derivatization reagents tested for the separation of peptides containing isomerized aspartic acid, 2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazine-2-yl) pyrrolidine-2-carboxylate (DMT-(R)-Pro-OSu) was the most suitable reagent for separating isomerized peptides and improved the sensitivity of mass spectrometry by 50-fold. This method was applied to analyze heat-denatured crystallin. Asp58 and Asp151 residues in αA-crystallin (AAC) exhibited the highest isomerization rate in heated crystallin. Furthermore, the analysis of α-crystallin extracted from bovine eye lens identified isomerized Asp residues (Asp24/35, Asp58, and Asp151 in AAC and Asp140 in αB-crystallin (ABC)). These results indicate that the newly developed method using chiral derivatization provides selective and sensitive analysis of isomerized Asp sites in α-crystallin protein. This novel method will allow for the identification and quantification of isomerized amino acids in crystallin proteins.


Assuntos
Ácido Aspártico/análise , Ácido Aspártico/química , Cromatografia Líquida/métodos , Cristalinas/química , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Humanos , Cristalino/química , Limite de Detecção , Peptídeos/química , Desnaturação Proteica , Estereoisomerismo
12.
Biochim Biophys Acta Proteins Proteom ; 1868(6): 140410, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32169581

RESUMO

Isomerization of aspartate (Asp) is a common non-enzymatic posttranslational modification. Isomerized residues accumulate in proteins associated with age-related human disorders such as cataract and are well known to affect protein structure and function. We previously detected d-Asp-containing peptides in human serum. In this study, we investigated whether isomerized Asp residues are present in human immunoglobulin G (IgG) kappa chain by a qualitative d-amino acid analysis based on diastereomer formation and liquid chromatography tandem mass spectrometry (LC-MS/MS). We also investigated the d/l ratio of Asp residues in the IgG kappa chain in serum from donors aged 25, 37, 41, 54 and 67 years. As a result, two isomerized Asp residues, Asp151 and Asp170, were detected in the IgG kappa chain, and the d/l ratio of these residues was found to increase with aging. To assess the effects of this isomerization, we synthesized four isomeric peptides of IgG kappa chain containing lα-, lß-, dα-, or dß-Asp at position 170, and compared their secondary structures by CD spectroscopy. Peptide containing normal lα-Asp170 showed type II ß-turn structure, while the other isomeric peptides showed random structure, clearly indicating that substitution of a single Asp isomer alters the secondary structure of the peptide. Because IgG is a main component of humoral immunity, Asp isomerization in IgG may reflect changes of structure and decrease in immune function. Proteome research on serum from the standpoint of racemization might enable us to develop new kinds of biomarker and new directions to study the aging process.


Assuntos
Envelhecimento/metabolismo , Ácido Aspártico/química , Imunoglobulina G/química , Fatores Imunológicos/química , Adulto , Idoso , Biomarcadores , Catarata/metabolismo , Cromatografia Líquida/métodos , Humanos , Imunoglobulina G/metabolismo , Cadeias kappa de Imunoglobulina , Isomerismo , Pessoa de Meia-Idade , Modelos Moleculares , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteoma , Soro , Estereoisomerismo , Espectrometria de Massas em Tandem/métodos
13.
Lett Appl Microbiol ; 70(5): 380-387, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32048328

RESUMO

Chitosan with higher molecular weight exhibited higher antimicrobial efficacy against foodborne pathogens. However, the poor water solubility of higher or medium molecular weight chitosan limits its applications. To overcome the challenge, our research team searched for simple preparation procedure for fast-dissolving medium molecular weight chitosan in water. Throughout the process, we were able to obtain a higher concentration of medium molecular weight water-soluble (MMWWS) chitosan (400 kDa). The MMWWS chitosan showed physicochemical properties that are suitable for edible coating. Antibacterial activities of 400-kDa chitosan coating prepared in acetic acid (1% v/v) or aspartic acid (1% or 3% w/v) were examined. The surface of catfish cubes was inoculated with six foodborne pathogens and then coated with chitosan solutions. The survival of each pathogen was evaluated during shelf life storage. Compared with the control, 3% w/v chitosan coating in aspartic acid solution exhibited the most effective antibacterial activities among other coating treatments, completely inhibiting Vibrio parahaemolyticus on the surface of catfish. The study suggested that chitosan dissolved in aspartic acid has the potential for use as an alternative antimicrobial coating for catfish fillet.


Assuntos
Anti-Infecciosos/farmacologia , Quitosana/farmacologia , Conservação de Alimentos/métodos , Ictaluridae/microbiologia , Animais , Anti-Infecciosos/química , Ácido Aspártico/química , Quitosana/química , Filmes Comestíveis , Microbiologia de Alimentos , Peso Molecular , Alimentos Marinhos/microbiologia
14.
Chem Commun (Camb) ; 56(20): 3047-3049, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32048688

RESUMO

A series of aminocarboxylic acid analogues of aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) were chemoenzymatically synthesized via the addition of various mono- and diamine substrates to fumaric acid catalyzed by the enzyme EDDS lyase. Many of these novel AMA and EDDS analogues demonstrate potent inhibition of the bacterial metallo-ß-lactamase NDM-1. Isothermal titration calorimetry assays revealed a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. Compounds 1a (AMA), 1b (AMB), 5 (EDDS), followed by 1d and 8a, demonstrate the highest synergy with meropenem resensitizing an NDM-1 producing strain of E. coli to this important carbapenem of last resort.


Assuntos
Ácido Aspártico/análogos & derivados , Complexos de Coordenação/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Etilenodiaminas/farmacologia , Succinatos/farmacologia , Zinco/farmacologia , Inibidores de beta-Lactamases/farmacologia , Aminoácidos/química , Aminoácidos/farmacologia , Ácido Aspártico/química , Ácido Aspártico/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Relação Dose-Resposta a Droga , Proteínas de Escherichia coli/metabolismo , Etilenodiaminas/química , Estrutura Molecular , Relação Estrutura-Atividade , Succinatos/química , Zinco/química , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismo
15.
Nat Commun ; 11(1): 982, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080186

RESUMO

Although peptide chemistry has made great progress, the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification or even inaccessible peptide sequences. Here, we report an alternative approach to address this longstanding challenge of peptide synthesis by utilizing cyanosulfurylides to mask carboxylic acids by a stable C-C bond. These functional groups-formally zwitterionic species-are exceptionally stable to all common manipulations and impart improved solubility during synthesis. Deprotection is readily and rapidly achieved under aqueous conditions with electrophilic halogenating agents via a highly selective C-C bond cleavage reaction. This protecting group is employed for the synthesis of a range of peptides and proteins including teduglutide, ubiquitin, and the low-density lipoprotein class A. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.


Assuntos
Peptídeos/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/síntese química , Ácido Aspártico/química , Ácidos Carboxílicos/química , Cianetos/química , Lipoproteínas LDL/síntese química , Lipoproteínas LDL/química , Dobramento de Proteína , Ubiquitina/química
16.
Phys Chem Chem Phys ; 22(5): 3008-3016, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957772

RESUMO

Infrared (IR) spectroscopy is commonly utilized for the investigation of protein structures and protein-mediated processes. While the amide I band provides information on protein secondary structures, amino acid side chains are used as IR probes for the investigation of protein reactions, such as proton pumping in rhodopsins. In this work, we calculate the IR spectra of the solvated aspartic acid, with both zwitterionic and protonated backbones, and of a capped form, i.e. mimicking the aspartic acid residue in proteins, by means of molecular dynamics (MD) simulations and the perturbed matrix method (PMM). This methodology has already proved its good modeling capabilities for the amide I mode and is here extended to the treatment of protein side chains. The computed side chain vibrational signal is in very good agreement with the experimental one, well reproducing both the peak frequency position and the bandwidth. In addition, the MD-PMM approach proposed here is able to reproduce the small frequency shift (5-10 cm-1) experimentally observed between the protonated and zwitterionic forms, showing that such a shift depends on the excitonic coupling between the modes localized on the side chain and on the backbone in the protonated form. The spectrum of the capped form, in which the amide I band is also calculated, agrees well with the corresponding experimental spectrum. The reliable calculation of the vibrational bands of carboxyl-containing side chains provides a useful tool for the interpretation of experimental spectra.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Proteínas/química , Espectrofotometria Infravermelho , Ácido Aspártico/química , Ácido Glutâmico/química , Teoria Quântica
17.
Biochemistry ; 59(7): 880-891, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31999436

RESUMO

Agonist binding to G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signaling pathways. Although high-resolution structures of different receptor states are available, atomistic details of allosteric signaling across the membrane remain elusive. We calculated free energy landscapes of ß2 adrenergic receptor activation using atomistic molecular dynamics simulations in an optimized string of swarms framework, which shed new light on how microswitches govern the equilibrium between conformational states. Contraction of the extracellular binding site in the presence of the agonist BI-167107 is obligatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. The connector is probabilistically coupled to the conformation of the intracellular region. An active connector promotes desolvation of a buried cavity, a twist of the conserved NPxxY motif, and an interaction between two conserved tyrosines in transmembrane helices 5 and 7 (Y-Y motif), which lead to a larger population of active-like states at the G protein binding site. This coupling is augmented by protonation of the strongly conserved Asp792.50. The agonist binding site hence communicates with the intracellular region via a cascade of locally connected microswitches. Characterization of these can be used to understand how ligands stabilize distinct receptor states and contribute to development drugs with specific signaling properties. The developed simulation protocol can likely be transferred to other class A GPCRs.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Benzoxazinas/química , Conformação Proteica/efeitos dos fármacos , Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Ácido Aspártico/química , Benzoxazinas/metabolismo , Sítios de Ligação , Humanos , Ligantes , Simulação de Dinâmica Molecular , Receptores Adrenérgicos beta 2/metabolismo , Sódio/química , Sódio/metabolismo , Termodinâmica
18.
Chem Commun (Camb) ; 56(10): 1537-1540, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922154

RESUMO

Although the underlying cause of Alzheimer's disease (AD) is not known, the extracellular deposition of ß-amyloid (Aß) is considered as a hallmark of AD brains. Evidence has shown the occurrence of d-Asp, isoAsp, and d-Ser residues in Aß, which may be indicative of and/or contribute to the neurodegeneration in AD patients. Herein, we have developed the first high-throughput profiling technique for all 20 isobaric Aß peptide epimers containing Asp, isoAsp, and Ser isomers using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This new analytical strategy allows the direct detection and identification of all possible Asp, isoAsp, and Ser stereoisomers in Aß, and may contribute to a better understanding of the pathogenesis of AD.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Cromatografia Líquida de Alta Pressão , Humanos , Serina/química , Estereoisomerismo , Espectrometria de Massas em Tandem
19.
Exp Eye Res ; 192: 107930, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31931001

RESUMO

The eye lens is mainly composed of crystallins, which undergo modifications such as oxidation, deamidation and isomerization with aging. Asp58, Asp76, Asp84, and Asp151 residues of αA-crystallin are site-specifically isomerized to L-iso, D-, and D-iso isomers in aged-related cataract lenses. In addition, an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human αA-crystallin, is detected in aged lens. This peptide induces protein aggregation and causes loss of the chaperone function of α-crystallin. The αA66-80 peptide contains Asp76, but it is not known whether isomerization of Asp76 in αA66-80 specifically induces protein aggregation or affects α-crystallin function. Using Fmoc-based solid-phase synthesis, here we synthesized four αA66-80 peptides, each containing L-, L-iso, D-, or D-isoAsp at position 76, and compared their structures and properties. Normal αA66-80 peptide containing the L-Asp76 isomer increased the EDTA-induced aggregation of ADH protein, DTT-induced aggregation of insulin, and heat-induced aggregation of ßL-crystallin. αA66-80 peptide containing D- or D-isoAsp76 had similar or no effects on the aggregation of these proteins. By contrast, αA66-80 peptide containing L-isoAsp76 inhibited the aggregation of all three proteins, indicating that it has chaperone activity. With regard to secondary structure, αA66-80 peptide containing the L-, D-, or D-isoAsp76 isomer had random-coil structure, whereas αA66-80 peptide containing L-isoAsp76 had ß-sheet like structure. A Thioflavin T (ThT) assay indicated that only the L-isoAsp-containing αA66-80 peptide has ß-sheet structure and generates amyloid fibrils. Collectively, these observations indicate that isomerization of Aps76 to the Lß isomer endows ß-sheet structure and chaperone function on this peptide.


Assuntos
Ácido Aspártico/química , Cristalino/química , Fragmentos de Peptídeos/química , Cadeia A de alfa-Cristalina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Bovinos , Cromatografia Líquida , Dicroísmo Circular , Isomerismo , Chaperonas Moleculares/química , Dados de Sequência Molecular , Agregação Patológica de Proteínas , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Biosci Biotechnol Biochem ; 84(3): 500-506, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31694479

RESUMO

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the separation and quantification of the enantiomers of N-methylaspartate and N-methylglutamate, after derivatization with Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide was established. The time required for the LC-ESI-MS/MS analysis was within 20 min and the detection limit was approximately 10 fmol per injection, demonstrating that this method can be used for the rapid determination of D-aspartate N-methyltransferase activity in the ark shell clam Scapharca broughtonii.Abbreviations: NMDA: N-methyl-D-aspartate; NMLA: N-methyl-L-aspartate; NMDG: N-methyl-D-glutamate; NMLG: N-methyl-L-glutamate; NMA: N-methylaspartate; NMG: N-methylglutamate; HPLC: high-performance liquid chromatography; SAM: S-adenosyl-L-methionine; OPA: o-phthalaldehyde; LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry; FDLA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide; FDAA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide; ESI: electrospray ionization; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry; MS/MS: tandem mass spectrometry.


Assuntos
Ácido Aspártico/química , Bivalves/metabolismo , Cromatografia Líquida/métodos , Metiltransferases/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Metiltransferases/química
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