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1.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639146

RESUMO

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9-30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9-30 is mainly disordered. We attempted to improve the affinity of HPr9-30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9-30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Mutação , Fragmentos de Peptídeos/farmacologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Histidina/química , Fragmentos de Peptídeos/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Staphylococcus aureus/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556577

RESUMO

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.


Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos/metabolismo , Elétrons , Heme/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Shewanella/metabolismo , Grupo dos Citocromos c/química , Citocromos/química , Transporte de Elétrons , Heme/química , Histidina/química , Metionina/química , Simulação de Dinâmica Molecular , Nanofios , Oxirredução
3.
DNA Cell Biol ; 40(10): 1325-1337, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34582699

RESUMO

Psoriasis is a chronic inflammatory skin disease characterized by massive keratinocyte proliferation and immune cell infiltration into the epidermis. However, the specific mechanisms underlying the development of psoriasis remain unclear. Untargeted metabolomics and transcriptomics have been used separately to profile biomarkers and risk genes in the serum of psoriasis patients. However, the integration of metabolomics and transcriptomics to identify dysregulated metabolites and genes in the psoriatic skin is lacking. In this study, we performed an untargeted metabolomics analysis of imiquimod (IMQ)-induced psoriasis-like mice and healthy controls, and found that levels of a total of 4,188 metabolites differed in IMQ-induced psoriasis-like mice compared with those in control mice. Metabolomic data analysis using MetaboAnalyst showed that the metabolic pathways of primary metabolites, such as folate biosynthesis and galactose metabolism, were significantly altered in the skin of mice after treatment with IMQ. Furthermore, IMQ treatment also significantly altered metabolic pathways of secondary metabolites, including histidine metabolism, in mouse skin tissues. The metabolomic results were verified by transcriptomics analysis. RNA-seq results showed that histamine decarboxylase (HDC) mRNA levels were significantly upregulated after IMQ treatment. Targeted inhibition of histamine biosynthesis process using HDC-specific inhibitor, pinocembrin (PINO), significantly alleviated epidermal thickness, downregulated the expression of interleukin (IL)-17A and IL-23, and inhibited the infiltration of immune cells during IMQ-induced psoriasis-like skin inflammation. In conclusion, our study offers a validated and comprehensive understanding of metabolism during the development of psoriasis and demonstrated that PINO could protect against IMQ-induced psoriasis-like skin inflammation.


Assuntos
Histidina/metabolismo , Metaboloma , Psoríase/metabolismo , Transcriptoma , Animais , Feminino , Imiquimode/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Psoríase/etiologia , Psoríase/genética
4.
Appl Environ Microbiol ; 87(20): e0133521, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34347519

RESUMO

ß-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of ß-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo-ß-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter-1) l-threo-ß-hydroxy-His and 150 mM l-threo-ß-hydroxy-Gln (24.3 g liter-1) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo-ß-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of ß-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for ß-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln with a titer of over 20 g liter-1 through whole-cell bioconversion using recombinant Escherichia coli cells. As ß-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , Glicina/metabolismo , Histidina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glicina/análogos & derivados , Histidina/análogos & derivados , Hidroxilação , Oxigenases de Função Mista/genética
5.
Nat Commun ; 12(1): 4845, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381036

RESUMO

The human gut microbiota is increasingly recognized as an important factor in modulating innate and adaptive immunity through release of ligands and metabolites that translocate into circulation. Urbanizing African populations harbor large intestinal diversity due to a range of lifestyles, providing the necessary variation to gauge immunomodulatory factors. Here, we uncover a gradient of intestinal microbial compositions from rural through urban Tanzanian, towards European samples, manifested both in relative abundance and genomic variation observed in stool metagenomics. The rural population shows increased Bacteroidetes, led by Prevotella copri, but also presence of fungi. Measured ex vivo cytokine responses were significantly associated with 34 immunomodulatory microbes, which have a larger impact on circulating metabolites than non-significant microbes. Pathway effects on cytokines, notably TNF-α and IFN-γ, differential metabolome analysis and enzyme copy number enrichment converge on histidine and arginine metabolism as potential immunomodulatory pathways mediated by Bifidobacterium longum and Akkermansia muciniphila.


Assuntos
Citocinas/imunologia , Microbioma Gastrointestinal/fisiologia , População Rural , População Urbana , Adulto , Arginina/metabolismo , Bactérias/imunologia , Bactérias/isolamento & purificação , Bactérias/metabolismo , Dieta , Feminino , Microbioma Gastrointestinal/imunologia , Histidina/metabolismo , Humanos , Imunomodulação , Masculino , Redes e Vias Metabólicas , Metaboloma/imunologia , Fatores Socioeconômicos , Tanzânia , Urbanização
6.
J Biol Chem ; 297(4): 101130, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461099

RESUMO

Cells synthesize proteins using 20 standard amino acids and expand their biochemical repertoire through intricate enzyme-mediated post-translational modifications (PTMs). PTMs can either be static and represent protein editing events or be dynamically regulated as a part of a cellular response to specific stimuli. Protein histidine methylation (Hme) was an elusive PTM for over 5 decades and has only recently attracted considerable attention through discoveries concerning its enzymology, extent, and function. Here, we review the status of the Hme field and discuss the implications of Hme in physiological and cellular processes. We also review the experimental toolbox for analysis of Hme and discuss the strengths and weaknesses of different experimental approaches. The findings discussed in this review demonstrate that Hme is widespread across cells and tissues and functionally regulates key cellular processes such as cytoskeletal dynamics and protein translation. Collectively, the findings discussed here showcase Hme as a regulator of key cellular functions and highlight the regulation of this modification as an emerging field of biological research.


Assuntos
Histidina/metabolismo , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Citoesqueleto/metabolismo , Humanos , Metilação
7.
PLoS One ; 16(7): e0253852, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34255797

RESUMO

Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Encéfalo/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Histidina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Metabolômica , Purinas/metabolismo , Pirimidinas/metabolismo , Ratos , Ratos Transgênicos
8.
FEBS Lett ; 595(16): 2113-2126, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245008

RESUMO

Histidine is an important carbon and nitrogen source of γ-proteobacteria and can affect bacteria-host interactions. The mechanisms of histidine uptake are only partly understood. Here, we analyze functional properties of the putative histidine transporter HutT of the soil bacterium Pseudomonas putida. The hutT gene is part of the histidine utilization operon, and the gene product belongs to the amino acid-polyamine-organocation (APC) family of secondary transporters. Deletion of hutT severely impairs growth of P. putida on histidine, suggesting that the encoded transporter is the major histidine uptake system of P. putida. Transport experiments with cells and purified and reconstituted protein indicate that HutT functions as a high-affinity histidine : proton symporter with high specificity for the amino acid. Substitution analyses identified amino acids crucial for HutT function.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Pseudomonas putida/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Pseudomonas putida/genética
9.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208680

RESUMO

In this paper, we present studies on the influence of the disulfide bridge on the copper (II) ions' binding abilities by the cyclic His4-peptide. The studied ligand HKHPHRHC-S-S-C consists of nine amino acids. The cyclic structure was obtained through a disulfide bridge between two cysteinyl groups. Moreover, this peptide is characterized by the presence of four His residues in the sequence, which makes it an interesting ligand for transition metal ions. The potentiometric and spectroscopic (UV-Vis spectroscopy and circular dichroism spectroscopy (CD)) studies were carried out in various molar ligand to metal ratios: 2:1, 1:1, and 1:2, in the pH range of 2.5-11 at 25 °C. The results showed that the cyclic His4-peptide promotes dinuclear complexes in each of these systems and forms the final dinuclear species with the {NIm, 3N-amide}{NIm, 3N-amide} coordination mode. The obtained data shows that cyclization by the formation of the disulfide bond has an impact on the peptide chain flexibility and appearance of additional potential donors for metal ions and influences the copper (II) ions' coordination.


Assuntos
Cobre/metabolismo , Histidina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cátions , Dissulfetos/química , Histidina/química , Concentração de Íons de Hidrogênio , Ligantes , Modelos Moleculares , Estrutura Molecular , Peptídeos Cíclicos , Ligação Proteica , Análise Espectral
10.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068879

RESUMO

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-ß peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aß sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu-Aß4-x] and [Cu-Aß1-x] complexes toward dopamine and other catechols. The results show that the CuII-ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [CuII-Aß-catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the CuI complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu-Aß4-x reveal that the His-tandem is able to bind CuII ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of CuII bound to the secondary site.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Complexos de Coordenação/metabolismo , Cobre/metabolismo , Dopamina/metabolismo , Oxirredutases/metabolismo , Peptídeos beta-Amiloides/química , Complexos de Coordenação/química , Cobre/química , Dopamina/química , Histidina/química , Histidina/metabolismo , Modelos Moleculares , Conformação Molecular , Oxirredução , Oxirredutases/química
11.
J Dairy Sci ; 104(9): 9917-9930, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34099295

RESUMO

The objective of this experiment was to determine the effect of increasing digestible His (dHis) levels with a rumen-protected (RP) His product on milk production, milk composition, and plasma AA concentrations in lactating dairy cows fed a metabolizable protein (MP)-deficient diet, according to the National Research Council dairy model from 2001. The companion paper presents results on the effect of increasing dHis dose with a MP-adequate basal diet. Twenty Holstein cows, of which 8 were rumen-cannulated, were used in a replicated 4 × 4 Latin square design experiment with four 28-d periods. Treatments were a control diet supplying 1.8% dHis of MP or 37 g/d (dHis1.8) and the control diet supplemented RP-His to provide 2.2, 2.6, or 3.0%, dHis of MP, or 53, 63, and 74 g/d (dHis2.2, dHis2.6, and dHis3.0, respectively). Histidine dose did not affect dry matter intake, but milk yield increased quadratically and energy-corrected milk yield increased linearly with increasing dHis dose. Histidine dose had a quadratic effect on milk fat concentration but did not affect milk fat yield. Lactose concentration decreased linearly, whereas lactose yield increased linearly with increasing dHis dose. There was a tendency for a linear increase in milk true protein concentration, and milk true protein yield increased linearly with dHis dose. Further, plasma His concentration increased linearly with increasing dHis dose and calculated apparent efficiency of His utilization decreased quadratically with increasing dHis supply. Histidine had minor or no effects on rumen fermentation. In the conditions of this experiment, RP-His supplementation of an MP-deficient corn silage-based diet increased milk yield linearly up to a dHis supply of 63 g/d (or 2.6% dHis of MP) and increased feed efficiency, energy-corrected milk yield and milk true protein yield linearly up to a dHis supply of 74 g/d (or 3.0% dHis of MP) in lactating dairy cows.


Assuntos
Histidina , Lactação , Ração Animal/análise , Animais , Bovinos , Dieta/veterinária , Feminino , Fermentação , Histidina/metabolismo , Leite/metabolismo , Proteínas do Leite/metabolismo , Rúmen/metabolismo
12.
Chem Commun (Camb) ; 57(36): 4460-4463, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33949502

RESUMO

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP.


Assuntos
Oxirredutases do Álcool/metabolismo , Escherichia coli/metabolismo , Compostos Férricos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lacase/metabolismo , Lipase/metabolismo , Oxirredutases do Álcool/química , Bacillus licheniformis/enzimologia , Bacillus subtilis/enzimologia , Candida tropicalis/enzimologia , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Compostos Férricos/química , Proteínas de Fluorescência Verde/química , Histidina/química , Histidina/metabolismo , Lacase/química , Lipase/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo
13.
Food Chem ; 358: 129884, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33933976

RESUMO

The ability of histidine to scavenge sugar-derived 1,2-dicarbonyl compounds was investigated using aqueous methanolic model systems containing histidine or histamine in the presence of glucose, methylglyoxal, or glyoxal. The samples were prepared either at room temperature (RT) or at 150 °C and analyzed using ESI-qTOF-MS/MS and isotope labeling technique. Replacing glucose with [U-13C6]glucose allowed the identification of glucose carbon atoms incorporated in the products. Various sugar-generated carbonyl compounds ranging in size from C1 to C6 were captured by histidine or histamine. The majority of the fragments incorporated were either C3 or C2 units originating from glyoxal (C2) or methylglyoxal (C3). The ESI-qTOF-MS/MS analysis indicated that histamine could react with either of the two carbonyl carbons of methylglyoxal utilizing the α-amino group and/or the imidazolium moiety. Furthermore, when histidine was added to 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) generating model system, it completely suppressed the formation of PhIP due to scavenging of phenylacetaldehyde.


Assuntos
Carnosina/química , Histamina/química , Histidina/química , Aldeído Pirúvico/química , Acetaldeído/análogos & derivados , Acetaldeído/química , Carnosina/metabolismo , Manipulação de Alimentos , Sequestradores de Radicais Livres/química , Glucose/química , Glioxal/química , Glioxal/metabolismo , Histidina/metabolismo , Imidazóis/química , Marcação por Isótopo , Aldeído Pirúvico/metabolismo , Açúcares/química , Espectrometria de Massas em Tandem , Temperatura
14.
PLoS One ; 16(5): e0251055, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33939760

RESUMO

Organs from donors after controlled circulatory death (DCD III) exhibit a higher risk for graft dysfunction due to an initial period of warm ischemia. This procurement condition can also affect the yield of beta cells in islet isolates from donor pancreases, and hence their use for transplantation. The present study uses data collected and generated by our Beta Cell Bank to compare the number of beta cells in isolates from DCD III (n = 141) with that from donors after brain death (DBD, n = 609), before and after culture, and examines the influence of donor and procurement variables. Beta cell number per DCD III-organ was significantly lower (58 x 106 versus 84 x 106 beta cells per DBD-organ; p < 0.001) but their purity (24% insulin positive cells) and insulin content (17 µg / 106 beta cells in DCD III-organs versus 19 µg / 106 beta cells in DBD-organs) were similar. Beta cell number correlated negatively with duration of acirculatory warm ischemia time above 10 min; for shorter acirculatory warm ischemia time, DCD III-organs did not exhibit a lower beta cell yield (74 x 106 beta cells). Use of Institut Georges Lopez-1 cold preservation solution instead of University of Wisconsin solution or histidine-tryptophan-ketoglutarate also protected against the loss in beta cell yield from DCD III-organs (86 x 106 for IGL-1 versus 54 x 106 and 65 x 106 beta cells respectively, p = 0.042). Multivariate analysis indicates that both limitation of acirculatory warm ischemia time and use of IGL-1 prevent the reduced beta cell yield in islet cell isolates from DCD III-organs.


Assuntos
Morte Encefálica/metabolismo , Morte Encefálica/patologia , Sobrevivência de Enxerto/fisiologia , Células Secretoras de Insulina/fisiologia , Soluções para Preservação de Órgãos/metabolismo , Adenosina/metabolismo , Adenosina/fisiologia , Adulto , Alopurinol/metabolismo , Feminino , Glutaratos/metabolismo , Glutationa/metabolismo , Glutationa/fisiologia , Histidina/metabolismo , Humanos , Insulina/metabolismo , Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Transplante de Fígado/métodos , Masculino , Pessoa de Meia-Idade , Rafinose/metabolismo , Rafinose/fisiologia , Doadores de Tecidos , Obtenção de Tecidos e Órgãos/métodos , Triptofano/metabolismo , Isquemia Quente/métodos
15.
Phys Chem Chem Phys ; 23(12): 7359-7367, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33876095

RESUMO

Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that bind a linear tetrapyrrole as a chromophore. They show photochromicity by having two stable states that can be interconverted by the photoisomerization of the chromophore. These photochemical properties make them an attractive target for biotechnological applications. However, their application is impeded by structural heterogeneity that reduces the yield of the photoconversion. The heterogeneity can originate either from the chromophore structure or the protein environment. Here, we study the origin of the heterogeneity in AnPixJg2, a representative member of the red/green cyanobacteriochrome family, that has a red absorbing parental state and a green absorbing photoproduct state. Using molecular dynamics simulations and umbrella sampling we have identified the protonation state of a conserved histidine residue as a trigger for structural heterogeneity. When the histidine is in a neutral form, the chromophore structure is homogenous, while in a positively charged form, the chromophore is heterogeneous with two different conformations. We have identified a correlation between the protonation of the histidine and the structural heterogeneity of the chromophore by detailed characterization of the interactions in the protein binding site. Our findings reconcile seemingly contradicting spectroscopic studies that attribute the heterogeneity to different sources. Furthermore, we predict that circular dichroism can be used as a diagnostic tool to distinguish different substates.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/química , Histidina/metabolismo , Proteínas de Bactérias/química , Cianobactérias/metabolismo , Teoria da Densidade Funcional , Histidina/química , Modelos Moleculares , Estrutura Molecular , Prótons
16.
Biochem J ; 478(9): 1795-1808, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33821889

RESUMO

To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.


Assuntos
Di-Hidroxifenilalanina/metabolismo , Heme/química , Histidina/metabolismo , Ferro/química , Mioglobina/metabolismo , Substituição de Aminoácidos , Biocatálise , Domínio Catalítico , Clonagem Molecular , Di-Hidroxifenilalanina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Histidina/genética , Humanos , Ligação de Hidrogênio , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Cinética , Modelos Moleculares , Mioglobina/química , Mioglobina/genética , Oxirredução , Peroxidases/química , Peroxidases/metabolismo , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
J Adv Res ; 29: 13-22, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33842001

RESUMO

Introduction: Intrinsic vitamin D affects the proliferation, apoptosis, invasion, metastasis, and tumorigenesis of lung cancer by regulating tumor signaling pathways. Histidine-rich calcium-binding protein (HRC) maintains Ca2+ homeostasis, which plays crucial roles in the occurrence and development of cancer. Objectives: Our study aims to investigate the ability of vitamin D in the regulation of HRC and the role of HRC playing in lung cancer. Methods: We investigated the effects of vitamin D on lung cancer and the underlying mechanisms, by measuring HRC and vitamin D receptor (VDR) expression in lung cancer, paracancer, and normal tissues from patients using immunohistochemistry, western blotting, and real time RT-PCR. We transfected H460 lung cancer cells (supplemented or not with vitamin D) with PX458-HRC and pcDNA3.1-HRC plasmids and injected mice with lung cancer cells harboring pcDNA3.1-vector or pcDNA3.1-HRC plasmids. Results: Vitamin D inhibited HRC expression and H460 cell migration and proliferation, and promoted apoptosis compared with controls. The expression of HRC and VDR was significantly upregulated and downregulated, respectively, in lung cancer versus paracancer or normal tissues. Cell proliferation and migration were reduced, apoptotic cells were more and tumors were smaller in mice treated with vitamin D/cholecalciferol cholesterol emulsion (CCE) than in vitamin D/CCE+HRC+/+ mice. Conclusion: Vitamin D inhibited lung cancer tumor growth, migration, and proliferation by downregulating HRC.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Vitamina D/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Histidina/metabolismo , Homeostase , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Calcitriol/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Vitaminas/farmacologia
18.
Phys Chem Chem Phys ; 23(17): 10475-10486, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33899866

RESUMO

The accumulation of ΔK280 tau mutant resulting in neurotoxic oligomeric aggregates is an important but yet mysterious procedure in Alzheimer's disease (AD) development. Recently, we proposed a histidine tautomerization hypothesis of tau fibrillogenesis for the pathobiology of AD and other neuro diseases. However, the influence of neutral histidine tautomeric states on tau mutation is still unclear. Herein, we performed replica-exchange molecular dynamics (REMD) simulations to characterize structural features as well as the mode of toxic action of the ΔK280 tau mutant in the presence of histidine tautomerism. Molecular dynamics (MD) simulation results show that the δε tautomeric isomer (having a distinct global energy minimum) had the highest ß-sheet structure, which adopts a sheet-rich conformer and may have significant influence on the structural behaviors of ΔK280 tau monomers. Furthermore, clustering, residual contact map, mobility and structural analysis exhibited that the presence of ß-strand interactions between stable lysine 8 (K8)-asparagine 13 (N13) and valine 39 (V39)-tyrosine 43 (Y43) residues plus K31-histidine 32 (H32) and K8-N13 (strand-loop-strand [ß-meander] structure) helped δε to form toxic aggregates. Moreover, H299 played a more critical role in the conformational instability of the δε than H268. Overall, the results obtained from this study may be used to arrest neurodegeneration in ΔK280 tau mutation carriers as well as increase the understanding of AD-related tau pathogenesis and strengthen the histidine tautomerism hypothesis of misfolded peptide accumulation.


Assuntos
Histidina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer , Histidina/química , Histidina/genética , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Simulação de Dinâmica Molecular , Mutação , Proteínas tau/química , Proteínas tau/genética
19.
FEBS Lett ; 595(12): 1708-1720, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33896006

RESUMO

The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His-brace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.


Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Cobre/química , Chaperonas Moleculares/química , Mutação de Sentido Incorreto , Pseudomonas fluorescens/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo
20.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33893237

RESUMO

Nonribosomal peptide synthetases (NRPSs) are large, multidomain biosynthetic enzymes involved in the assembly-line-like synthesis of numerous peptide natural products. Among these are clinically useful antibiotics including three classes of ß-lactams: the penicillins/cephalosporins, the monobactams, and the monocyclic nocardicins, as well as the vancomycin family of glycopeptides and the depsipeptide daptomycin. During NRPS synthesis, peptide bond formation is catalyzed by condensation (C) domains, which couple the nascent peptide with the next programmed amino acid of the sequence. A growing number of additional functions are linked to the activity of C domains. In the biosynthesis of the nocardicins, a specialized C domain prepares the embedded ß-lactam ring from a serine residue. Here, we examine the evolutionary descent of this unique ß-lactam-synthesizing C domain. Guided by its ancestry, we predict and demonstrate in vitro that this C domain alternatively performs peptide bond formation when a single stereochemical change is introduced into its peptide starting material. Remarkably, the function of the downstream thioesterase (TE) domain also changes. Natively, the TE directs C terminus epimerization prior to hydrolysis when the ß-lactam is made but catalyzes immediate release of the alternative peptide. In addition, we investigate the roles of C-domain histidine residues in light of clade-specific sequence motifs, refining earlier mechanistic proposals of both ß-lactam formation and canonical peptide synthesis. Finally, expanded phylogenetic analysis reveals unifying connections between ß-lactam synthesis and allied C domains associated with the appearance of ᴅ-amino acid and dehydroamino acid residues in other NRPS-derived natural products.


Assuntos
Antibacterianos/biossíntese , Evolução Molecular , Lactamas/metabolismo , Peptídeo Sintases/genética , Histidina/metabolismo , Peptídeo Sintases/metabolismo , Tioléster Hidrolases/metabolismo
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