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1.
Nat Commun ; 11(1): 4628, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934220

RESUMO

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.


Assuntos
Arginina/química , Lisina/química , Cinética , Transição de Fase , Polinucleotídeos/química , Viscosidade
2.
Int J Nanomedicine ; 15: 3639-3647, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547019

RESUMO

Purpose: Astrocyte dysfunction is a hallmark of central nervous system injury or infection. As a primary contributor to neurodegeneration, astrocytes are an ideal therapeutic target to combat neurodegenerative conditions. Gene therapy has arisen as an innovative technique that provides excellent prospect for disease intervention. Poly (lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) are polymeric nanoparticles commonly used in gene delivery, each manifesting their own set of advantages and disadvantages. As a clinically approved polymer by the Federal Drug Administration, well characterized for its biodegradability and biocompatibility, PLGA-based nanoparticles (PLGA-NPs) are appealing for translational gene delivery systems. However, our investigations revealed PLGA-NPs were ineffective at facilitating exogenous gene expression in primary human astrocytes, despite their success in other cell lines. Furthermore, PEI polymers illustrate high delivery efficiency but induce cytotoxicity. The purpose of this study is to develop viable and biocompatible NPsystem for astrocyte-targeted gene therapy. Materials and Methods: Successful gene expression by PLGA-NPs alone or in combination with arginine-modified PEI polymers (AnPn) was assessed by a luciferase reporter gene encapsulated in PLGA-NPs. Cytoplasmic release and nuclear localization of DNA were investigated using fluorescent confocal imaging with YOYO-labeled plasmid DNA (pDNA). NP-mediated cytotoxicity was assessed via lactate dehydrogenase in primary human astrocytes and neurons. Results: Confocal imaging of YOYO-labeled pDNA confirmed PLGA-NPs delivered pDNA to the cytoplasm in a dose and time-dependent manner. However, co-staining revealed pDNA delivered by PLGA-NPs did not localize to the nucleus. The addition of AnPn significantly improved nuclear localization of pDNA and successfully achieved gene expression in primary human astrocytes. Moreover, these formulations were biocompatible with both astrocytes and neurons. Conclusion: By co-transfecting two polymeric NPs, we developed an improved system for gene delivery and expression in primary human astrocytes. These findings provide a basis for a biocompatible and clinically translatable method to regulate astrocyte function during neurodegenerative diseases and disorders.


Assuntos
Arginina/química , Astrócitos/metabolismo , Técnicas de Transferência de Genes , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , DNA/genética , Células HEK293 , Humanos , Tamanho da Partícula , Plasmídeos/genética , Polietilenoimina , Transfecção
3.
Proc Natl Acad Sci U S A ; 117(27): 15731-15739, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561643

RESUMO

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.


Assuntos
Arginina/química , Nucleoproteínas/genética , Ornitina/química , Peptídeos/genética , Sequência de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Arginina/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleoproteínas/química , Ornitina/genética , Peptídeos/química , Proteínas/química , Proteínas/genética , RNA/química , RNA/genética
4.
Food Chem ; 327: 127039, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454273

RESUMO

In this study, we investigated the tailoring of food emulsions using interactions between rice bran cellulose nanocrystals (CNCs) and lauric arginate (LAE), which is food-grade cationic surfactant. Complexes of anionic CNCs and cationic LAE (CNCs/LAE) were formed through electrostatic attraction which were characterized using isothermal titration calorimetry (ITC), turbidity, and zeta-potential measurements. The saturation complexes could be formed at ratios of 1:2 (w/w) CNCs-to-LAE. Furthermore, the physical and oxidative stability of oil-in-water emulsions containing lipid droplets coated by CNCs/LAE complexes was determined. Electrostatic complexes formed from 0.02% CNCs and 0.1% LAE produced stable Pickering emulsions that were resistant to droplet coalescence. It was also exhibited that 0.02% CNCs and 0.1% LAE complexes stabilized-emulsions was able to extend the lag phase to 20 days for lipid hydroperoxide and to 14 days for hexanal production. This study shows that food-grade Pickering emulsions with good stability can be produced by CNCs with LAE complexes.


Assuntos
Arginina/análogos & derivados , Celulose/química , Alimentos , Nanopartículas/química , Óleos/química , Tensoativos/química , Água/química , Arginina/química , Emulsões , Eletricidade Estática
5.
Proc Natl Acad Sci U S A ; 117(21): 11421-11431, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393642

RESUMO

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas Intrinsicamente Desordenadas/química , Substituição de Aminoácidos , Arginina/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Microrganismos Geneticamente Modificados , Simulação de Dinâmica Molecular , Transição de Fase , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Tirosina/química
6.
Int J Nanomedicine ; 15: 1837-1851, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32256063

RESUMO

Introduction: Gold nanorods are highly reactive, have a large surface-to-volume ratio, and can be functionalized with biomolecules. Gold nanorods can absorb infrared electromagnetic radiation, which is subsequently dispersed as local heat. Gold nanoparticles can be used as powerful tools for the diagnosis and therapy of different diseases. To improve the biological barrier permeation of nanoparticles with low cytotoxicity, in this study, we conjugated gold nanorods with cell-penetrating peptides (oligoarginines) and with the amphipathic peptide CLPFFD. Methods: We studied the interaction of the functionalized gold nanorods with biological membrane models (liposomes) by dynamic light scattering, transmission electron microscopy and the Langmuir balance. Furthermore, we evaluated the effects on cell viability and permeability with an MTS assay and TEM. Results and Discussion: The interaction study by DLS, the Langmuir balance and cryo-TEM support that GNR-Arg7CLPFFD enhances the interactions between GNRs and biological membranes. In addition, cells treated with GNR-Arg7CLPFFD internalized 80% more nanoparticles than cells treated with GNR alone and did not induce cell damage. Conclusion: Our results indicate that incorporation of an amphipathic sequence into oligoarginines for the functionalization of gold nanorods enhances biological membrane nanoparticle interactions and nanoparticle cell permeability with respect to nanorods functionalized with oligoarginine. Overall, functionalized gold nanorods with amphipathic arginine rich peptides might be candidates for improving drug delivery by facilitating biological barrier permeation.


Assuntos
Peptídeos Penetradores de Células/química , Lipossomos/farmacocinética , Nanotubos/química , Arginina/química , Linhagem Celular Tumoral , Sobrevivência Celular , Peptídeos Penetradores de Células/farmacocinética , Sistemas de Liberação de Medicamentos , Difusão Dinâmica da Luz , Ouro/química , Humanos , Lipossomos/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Peptídeos/química
7.
Proc Natl Acad Sci U S A ; 117(15): 8503-8514, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234784

RESUMO

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/química
8.
Arch Biochem Biophys ; 686: 108373, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32325089

RESUMO

Non-enzymatic protein glycation results in the formation of advanced glycation end products (AGEs) leads to the pathogenesis of long-term diabetic complications. Iridin (ID), an antioxidant, plays an important role in protecting against oxidative stress and could therefore be an efficacious anti-glycating regimen. Herein, we assessed the anti-glycating potential of ID against d-ribose induced glycation of bovine serum albumin (BSA) by various biophysical and biochemical techniques. Our results from several physicochemical assays advocated that ID was able to evidently prevent the AGEs generation via reducing hyperchromicity, early glycation products (EGPs), carbonyl content (CC), hydroxymethyl furfural (HMF) content, production of fluorescent AGEs, protection against loss of secondary structure (i.e. α-helix and ß-sheets) of proteins, increasing the free lysine and free arginine content, reduced binding of congo red (CR), and reduced thioflavin T (ThT) and 8-aninilo-1-napthalene sulphonate (ANS)-specific fuorescence in glycated-BSA (Gly-BSA). On the basis of these findings, we concluded that ID possesses the significant anti-glycation potential and may be established as a remarkable anti-AGEs therapeutic agent. Further in-vivo and clinical studies are still warranted to uncover the therapeutic effects of ID against age-related as well as metabolic diseases.


Assuntos
Antioxidantes/química , Protaminas/química , Ribose/química , Soroalbumina Bovina/química , Arginina/química , Benzotiazóis/química , Sítios de Ligação , Corantes Fluorescentes/química , Produtos Finais de Glicação Avançada/química , Glicosilação , Lisina/química , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Estrutura Secundária de Proteína
9.
Nat Chem Biol ; 16(5): 587-595, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32123387

RESUMO

The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.


Assuntos
Arginina/química , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/metabolismo , Reparo de Erro de Pareamento de DNA , Escherichia coli/genética , Células HEK293 , Humanos , Células MCF-7 , Conformação Proteica , RNA/metabolismo
10.
Food Chem ; 320: 126619, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32203836

RESUMO

The fermentation of mare's milk into koumiss produces many beneficial functional compounds depending on the metabolism of the initial microbial flora. In this study, metabolites found in mare's milk and resulting koumiss were identified. Major metabolic pathways in the fermentation were also identified using an UPLC-Q-TOF-MS-based metabolomics method. In total, 354 metabolites were identified: 61 were up-regulated and 105 were down-regulated. Metabolic pathway analyses revealed that c-5-branched dibasic acid metabolism, valine, leucine and isoleucine degradation, arginine and proline metabolism, valine, leucine and isoleucine biosynthesis, vascular smooth muscle contraction, aminoacyl-tRNA biosynthesis and ß-alanine metabolism showed significant increases. A hierarchical cluster analysis of metabolites indicated a clear grouping pattern in which the relative concentrations of p-pyruvate, 20-HETE, 4-aminobutanoate, uracil, acetoacetate, and γ-linolenic acid differed significantly between milk and koumiss. This study provides reference values for metabolic isolates and bioactive compounds purification in mare's milk and koumiss.


Assuntos
Kumis/análise , Metabolômica , Leite/química , Animais , Arginina/química , Arginina/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Fermentação , Cavalos , Leucina/química , Leucina/metabolismo , Espectrometria de Massas , Leite/metabolismo , Valina/química , Valina/metabolismo
11.
Food Chem ; 318: 126516, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32146313

RESUMO

This work investigated the effects of L-arginine (Arg) and L-lysine (Lys) on the tenderness of chicken breast and explored the possible mechanisms underlying this effect for the first time. The results showed that both Arg and Lys decreased the shear force and increased the pH value, sarcomere length and myofibrillar fragmentation index as well as degraded the troponin-T by keeping calpain activity in chicken breast. In addition, Arg effectively reduced Ca2+/Mg2+-ATPase activities and promoted actomyosin dissociation. These results indicated that both Arg and Lys could enhance the tenderness of chicken breast, and it could also explain why Arg was more effective than Lys in improving the tenderness of chicken breast. These results will help facilitate the development of industrial-scale methods for improving the tenderness of meat products.


Assuntos
Actomiosina/química , Arginina/farmacologia , Galinhas , Lisina/farmacologia , Produtos Avícolas , Troponina T/química , Animais , Arginina/química , Calpaína/química , Calpaína/metabolismo , Qualidade dos Alimentos , Concentração de Íons de Hidrogênio , Lisina/química
12.
Biochem Pharmacol ; 174: 113850, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32044355

RESUMO

The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1.


Assuntos
Arginina/metabolismo , Asparagina/metabolismo , Simulação por Computador , Família 4 do Citocromo P450/metabolismo , Arginina/química , Arginina/genética , Asparagina/química , Asparagina/genética , Sítios de Ligação/fisiologia , Família 4 do Citocromo P450/química , Família 4 do Citocromo P450/genética , Humanos , Mutação de Sentido Incorreto/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato/fisiologia
13.
J Biomol NMR ; 74(2-3): 173-181, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32008172

RESUMO

Arginine side chains play critical roles in many protein-ligand interactions and enzyme catalysis. Unambiguous resonance assignment is a prerequisite for the nuclear magnetic resonance (NMR) spectroscopy studies of arginine side chains dynamics and hydrogen exchange properties from which one can expect to elucidate in more detail the roles of arginine residues in protein structure and function. Here we present a new mass spectrometry (MS)-based method for assigning the side-chain resonances of arginine residues in 2D 1H-15N NMR spectra. The method requires no additional isotopic labeling, and relies on knowledge of the amino acid sequence, the modification of the guanidino groups and liquid chromatography-mass spectrometry rather than the protein's structure or properties. Correlating the modification rates can connect cross-peak positions from NMR data with MS data to support resonances assignments. In the present work, we have extended our original application to natural abundance human ubiquitin to provide ε-NH assessments of three arginine for this well-studied protein.


Assuntos
Arginina/química , Marcação por Isótopo , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular , Ubiquitina/química , Cromatografia Líquida , Humanos
14.
Biochemistry ; 59(8): 943-954, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32031785

RESUMO

The projected decline of available phosphorus necessitates alternative methods to derive usable phosphate for fertilizer and other applications. Phosphite dehydrogenase oxidizes phosphite to phosphate with the cofactor NAD+ serving as the hydride acceptor. In addition to producing phosphate, this enzyme plays an important role in NADH cofactor regeneration processes. Mixed quantum mechanical/molecular mechanical free energy simulations were performed to elucidate the mechanism of this enzyme and to identify the protonation states of the substrate and product. Specifically, the finite temperature string method with umbrella sampling was used to generate the free energy surfaces and determine the minimum free energy paths for six different initial conditions that varied in the protonation state of the substrate and the position of the nucleophilic water molecule. In contrast to previous studies, the mechanism predicted by all six independent strings is a concerted but asynchronous dissociative mechanism in which hydride transfer from the phosphite substrate to NAD+ occurs prior to attack by the nucleophilic water molecule. His292 is identified as the most likely general base that deprotonates the attacking water molecule. However, Arg237 could also serve as this base if it were deprotonated and His292 were protonated prior to the main chemical transformation, although this scenario is less probable. The simulations indicate that the phosphite substrate is monoanionic in its active form and that the most likely product is dihydrogen phosphate. These mechanistic insights may be helpful for designing mutant enzymes or artificial constructs that convert phosphite to phosphate and NAD+ to NADH more effectively.


Assuntos
NADH NADPH Oxirredutases/química , Arginina/química , Teoria da Densidade Funcional , Histidina/química , Modelos Químicos , NAD/química , Fosfitos/química , Pseudomonas stutzeri/enzimologia , Termodinâmica , Água/química
15.
Artigo em Inglês | MEDLINE | ID: mdl-32062367

RESUMO

Protein-arginine methyltransferases catalyze the methylation of the guanidine (NG) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of NG-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous NG-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH2), asymmetrically NG-dimethylated vasopressin (cV-ADMA-Gly-NH2) and symmetrically NG-dimethylated vasopressin (cV-SDMA-Gly-NH2) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 were discriminated from cV-Arg-Gly-NH2 by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH2, oV-SDMA-Gly-NH2 and oV-Arg-Gly-NH2. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M-H)]+ and [(M-H)+H]+ in the positive ESI mode and undergo in part conversion of their terminal Gly-NH2 (NH2, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH2. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic NG-dimethylated proteins, including spectrin-α (280 kDa), spectrin-ß (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for NG-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.


Assuntos
Arginina Vasopressina , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Guanidina/química , Espectrometria de Massas em Tandem/métodos , Arginina/análogos & derivados , Arginina/análise , Arginina/sangue , Arginina/química , Arginina Vasopressina/análise , Arginina Vasopressina/sangue , Arginina Vasopressina/química , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Humanos , Modelos Lineares , Masculino , Peptídeos/análise , Peptídeos/sangue , Peptídeos/química , Projetos Piloto , Processamento de Proteína Pós-Traducional
16.
J Biol Chem ; 295(7): 2113-2124, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31914412

RESUMO

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn71, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.


Assuntos
Catálise , Hidrolases/ultraestrutura , Conformação Proteica , Synechocystis/ultraestrutura , Amônia/química , Arginina/química , Dióxido de Carbono/metabolismo , Cristalografia por Raios X , Hidrolases/química , Hidrolases/genética , Nitrogênio/química , Ornitina/química , Synechocystis/enzimologia
17.
Nat Commun ; 11(1): 571, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996674

RESUMO

Aggregation of the Tau protein into fibrils defines progression of neurodegenerative diseases, including Alzheimer's Disease. The molecular basis for potentially toxic reactions of Tau aggregates is poorly understood. Here we show that π-stacking by Arginine side-chains drives protein binding to Tau fibrils. We mapped an aggregation-dependent interaction pattern of Tau. Fibrils recruit specifically aberrant interactors characterised by intrinsically disordered regions of atypical sequence features. Arginine residues are key to initiate these aberrant interactions. Crucial for scavenging is the guanidinium group of its side chain, not its charge, indicating a key role of π-stacking chemistry for driving aberrant fibril interactions. Remarkably, despite the non-hydrophobic interaction mode, the molecular chaperone Hsp90 can modulate aberrant fibril binding. Together, our data present a molecular mode of action for derailment of protein-protein interaction by neurotoxic fibrils.


Assuntos
Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Arginina/metabolismo , Ligação Proteica , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Arginina/química , Progressão da Doença , Guanidina/metabolismo , Proteínas de Choque Térmico HSP90 , Humanos , Espectrometria de Massas , Chaperonas Moleculares , Agregados Proteicos , Domínios Proteicos , Dobramento de Proteína , Proteoma , Ratos , Análise de Sequência de Proteína , Proteínas tau/química , Proteínas tau/genética
18.
J Chromatogr A ; 1618: 460890, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31980261

RESUMO

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.


Assuntos
Arginina/química , Técnicas de Química Analítica/métodos , Cromatografia de Afinidade , DNA Super-Helicoidal/isolamento & purificação , Plasmídeos/isolamento & purificação , Proteína Supressora de Tumor p53/genética , Sefarose/química
19.
Food Chem ; 312: 126122, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31901825

RESUMO

This study investigated how l-lysine/l-arginine/l-cysteine improved cured sausage color. Visible detection showed that Mb(Fe2+)NO did not form when MetMb(Fe3+) was treated with a combination of l-lysine and NaNO2, l-arginine and NaNO2, or l-cysteine and NaNO2. Visible spectra and electron paramagnetic resonance (EPR) detection together indicated formation of Mb(Fe2+)O2 when MetMb(Fe3+) was treated with l-lysine, l-arginine, or l-cysteine; Mb(Fe2+)NO was formed when MetMb(Fe3+) was treated with a combination of l-lysine and NO, l-arginine and NO, or l-cysteine and NO. Visible, EPR, and fourier transform infrared results suggested formation of a complex of Mb(Fe2+) and l-cysteine by the coordination of sulfydryl and ferrous porphyrin. Therefore, l-lysine, l-arginine, or l-cysteine can promote the formation of Mb(Fe2+)NO by reducing MetMb(Fe3+) to Mb(Fe2+)O2, and l-cysteine promotes formation of a complex of Mb(Fe2+) and l-cysteine via coordination of sulfydryl and ferrous porphyrin, which may improve cured sausage color.


Assuntos
Produtos da Carne/análise , Arginina/química , Cor , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Lisina/química
20.
Int J Food Microbiol ; 320: 108519, 2020 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31962221

RESUMO

A novel composite antimicrobial film (CAF), made from a pullulan-based biopolymer and polyethylene (PE) was developed and evaluated for controlling pathogens associated with muscle foods. Initially, CAFs were developed by incorporating thymol (T), nisin (N) and/or lauric arginate (LAE) into the pullulan layer and layering it on top of PE. The antimicrobial activity of the resulting CAFs was evaluated against cocktails of Shiga toxin-producing E. coli (STEC), Salmonella spp., Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) in disk diffusion assays (DDAs). CAFs containing N were ineffective, while those containing T were effective for inhibiting the pathogens in DDAs. However, CAFs made with them did not exhibit desirable physical and mechanical properties since solvents (HCl and ethanol, respectively) interfered with the binding of pullulan to PE. Conversely, CAFs made with 0.5, 1 and 2.5% LAE maintained proper physical and mechanical characteristics and inhibited the four bacterial pathogens in DDAs. Based on these preliminary results, cocktails consisting of approximately 8 log10 CFU/ml of STEC, Salmonella, L. monocytogenes, or S. aureus were experimentally-inoculated onto raw beef, raw chicken breast, or ready-to-eat (RTE) turkey breast to obtain approximately 6.6 log10 CFU/cm2, aseptically transferred to CAFs containing 0.5, 1, or 2.5% LAE that were made into sachets/bags, vacuum packaged, sealed, and remaining microbial populations determined up to 28 days of refrigerated storage (4 °C). By day 28, CAFs containing 0.5, 1, and 2.5% LAE reduced: STEC by 1.13, 1.33 and 2.88 log10 CFU/cm2 respectively, on raw beef; Salmonella by 2.03, 2.12 and 3.01 log10 CFU/cm2 respectively, on raw chicken breast; L. monocytogenes by 1.12, 1.81 and 3.56 log10 CFU/cm2 respectively, on RTE turkey breast; and S. aureus by 0.68, 2.02 and 3.43 log10 CFU/cm2, respectively, on RTE turkey breast. CAFs may be of interest to the meat and poultry industry to control foodborne pathogens associated with these food products.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Embalagem de Alimentos/instrumentação , Carne/microbiologia , Animais , Arginina/análogos & derivados , Arginina/química , Arginina/farmacologia , Bactérias/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Embalagem de Alimentos/métodos , Glucanos/química , Glucanos/farmacologia , Nisina/química , Nisina/farmacologia , Timol/química , Timol/farmacologia
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