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1.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3348-3360, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622641

RESUMO

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.


Assuntos
Mirabilis , Saccharomyces cerevisiae , Flavobacterium , Ensaios de Triagem em Larga Escala , Saccharomyces cerevisiae/genética , Tirosina
2.
Nature ; 597(7876): 420-425, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471290

RESUMO

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Ácidos Mandélicos/metabolismo , Metaboloma , Ubiquinona/análogos & derivados , Animais , Linhagem Celular , Feminino , Humanos , Ácidos Mandélicos/análise , Camundongos , Camundongos Nus , Tirosina/metabolismo , Ubiquinona/biossíntese
3.
Langmuir ; 37(37): 10883-10889, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34498463

RESUMO

In living organisms, tyrosinase selectively produces l-DOPA from l-tyrosine. Here, a bicomponent hydrogel is used as a template for tyrosinase-catalyzed selective generation of l-DOPA from tyrosine. An amphiphilic molecule 1,5-diaminonaphthalene (DAN) coassembles with 1,3,5-benzenetricarboxylic acid (BTC) to form a self-supporting hydrogel. After alteration of complementary acids, DAN does not coassemble to form a hydrogel. The coassembly mechanism is investigated using spectroscopic techniques. The transmission electron microscopy and scanning electron microscopy images reveal the morphology details. The l-DOPA is kept from being oxidized when the hydrogel is used as a template. The enzymatically synthesized l-DOPA can also be separated from the mixture by easy tuning of the bicomponent coassembly.


Assuntos
Hidrogéis , Monofenol Mono-Oxigenase , Levodopa , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Tirosina
4.
Phys Chem Chem Phys ; 23(34): 18525-18534, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34581329

RESUMO

The ultrafast dynamics triggered by the photodetachment of the tyrosinate dianion in aqueous environment shed light on the elementary processes that accompany the interaction of ionizing radiation with biological matter. Photodetachment of the tryosinate dianion yields the tyrosyl radical anion, an important intermediate in biological redox reactions, although the study of its ultrafast dynamics is limited. Here, we utilize femtosecond optical pump-probe spectroscopy to investigate the ultrafast structural reorganization dynamics that follow the photodetachment of the tyrosinate dianion in aqueous solution. Photodetachment of the tyrosinate dianion leads to vibrational wave packet motion along seven vibrational modes that are coupled to the photodetachment process. The vibrational modes are assigned with the aid of density functional theory (DFT) calculations. Our results offer a glimpse of the elementary dynamics of ionized biomolecules and suggest the possibility of extending this approach to investigate the ionization-induced structural rearrangement of other aromatic amino acids and larger biomolecules.


Assuntos
Radicais Livres/química , Tirosina/química , Ânions/química , Teoria da Densidade Funcional , Hidróxido de Sódio/química , Espectrofotometria , Água/química
5.
Enzyme Microb Technol ; 150: 109884, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489037

RESUMO

Tyrosinase plays an essential role in melanin biosynthesis and inherently exhibits both monophenolase and diphenolase activity. A first derivative synchronous fluorometric assay was established for directly monitoring monophenolase activity. The zero-crossing point at 322 nm for the first-derivative under synchronous fluorescence with Δλ = 67 nm was utilized to selectively quantify tyrosine in the presence of the reaction product dihydroxyphenylalanine (DOPA). The limit of detection (LOD) for tyrosine was 0.54 µM. The fluorescence intensity of tyrosine was monitored at intervals of 30 s to establish the time course of tyrosine consumption. The LOD for the monophenolase activity was 0.0706 U⋅ mL-1. The Michaelis-Menten e constant and maximum speed were 21.83 µM and 1.12 µM min-1, respectively. Zinc ions competitively inhibited the monophenolase activity, with an IC50 value of 14.36 µM. This assay is easily and rapidly executed and is of great significance for analyzing the kinetics of enzymatic reactions and in fundamental research on monophenolase. This approach has potential applications in the discovery of tyrosinase inhibitors for medicine and cosmetics, as well as in the industrial synthesis of substituted o-diphenol intermediates.


Assuntos
Monofenol Mono-Oxigenase , Oxirredutases , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Oxirredutases/metabolismo , Tirosina/metabolismo
6.
Croat Med J ; 62(4): 310-317, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34472733

RESUMO

AIM: To investigate the diagnostic accuracy of O-(2-[18F]-fluoroethyl)-L-tyrosine (18F-FET) and fluoromethyl-(18F)-dimethyl-2-hydroxyethyl-ammonium chloride (18F-FCH) computed tomography (CT) in patients with primary low-grade gliomas (LGG). METHODS: The study enrolled patients with magnetic resonance imaging (MRI)-suspected LGG. Patients underwent both 18F-FET and 18F-FCH positron emission tomography (PET)-CT. Brain PET-CT was performed according to standard protocol - 20 minutes after intravenous injection of 185 MBq of 18F-FET and 185 MBq of 18F-FCH PET. Surgery and pathohistological diagnosis were performed in the next two weeks. RESULTS: We observed significantly better concordance between tumor histology and 18F-FET PET (weighted Kappa 0.74) compared with both 18F-FCH (weighted Kappa 0.15) and MRI (weighted Kappa 0.00). Tumor histology was significantly associated with 18F-FET (odds ratio 12.87; 95% confidence interval [CI], 0.49-333.70; P=0.013, logistic regression analysis). Receiver operating characteristic curve analysis comparing 18F-FCH (area under the curve [AUC] 0.625, 95% CI 0.298-0.884) and 18F-FET (AUC 0.833, 95% CI 0.499-0.982) showed better diagnostic properties of 18F-FET (AUC difference 0.208, 95% CI -0.145 to 0.562, P=0.248). CONCLUSION: Performing PET-CT in patients with newly diagnosed LGG should be preceded by a selection of an appropriate radiopharmaceutical. 18F-FET seems to be more accurate than 18F-FCH in the LGG diagnosis.


Assuntos
Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/diagnóstico por imagem , Colina/análogos & derivados , Glioma/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Projetos Piloto , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Tirosina
7.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445381

RESUMO

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.


Assuntos
Hemina/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Lacase/metabolismo , Albumina Sérica Humana/química , Tirosina/química , Sítios de Ligação , Biocatálise , Química Click , Sistemas de Liberação de Medicamentos , Humanos , Ibuprofeno/química , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Albumina Sérica Humana/metabolismo
8.
Inorg Chem ; 60(17): 13539-13549, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34382397

RESUMO

The peroxide-dependent coproheme decarboxylase ChdC from Geobacillus stearothermophilus catalyzes two key steps in the synthesis of heme b, i.e., two sequential oxidative decarboxylations of coproporphyrinogen III (coproheme III) at propionate groups P2 and P4. In the binding site of coproheme III, P2 and P4 are anchored by different residues (Tyr144, Arg217, and Ser222 for P2 and Tyr113, Lys148, and Trp156 for P4); however, strong experimental evidence supports that the generated Tyr144 radical acts as an unique intermediary for hydrogen atom transfer (HAT) from both reactive propionates. So far, the reaction details are still unclear. Herein, we carried out quantum mechanics/molecular mechanics calculations to explore the decarboxylation mechanism of coproheme III. In our calculations, the coproheme Cpd I, Fe(IV) = O coupled to a porphyrin radical cation (por•+) with four propionate groups, was used as a reactant model. Our calculations reveal that Tyr144 is directly involved in the decarboxylation of propionate group P2. First, the proton-coupled electron transfer (PCET) occurs from Tyr144 to P2, generating a Tyr144 radical, which then abstracts a hydrogen atom from the Cß of P2. The ß-H extraction was calculated to be the rate-limiting step of decarboxylation. It is the porphyrin radical cation (por•+) that makes the PCET from Tyr144 to P2 to be quite easy to initiate the decarboxylation. Finally, the electron transfers from the Cß• through the porphyrin to the iron center, leading to the decarboxylation of P2. Importantly, the decarboxylation of P4 mediated by Lys148 was calculated to be very difficult, which suggests that after the P2 decarboxylation, the generated harderoheme III intermediate should rebind or rotate in the active site so that the propionate P4 occupies the binding site of P2, and Tyr144 again mediates the decarboxylation of P4. Thus, our calculations support the fact that Tyr144 is responsible for the decarboxylation of both P2 and P4.


Assuntos
Proteínas de Bactérias/química , Carboxiliases/química , Coproporfirinogênios/química , Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Domínio Catalítico , Coproporfirinogênios/metabolismo , Descarboxilação , Elétrons , Geobacillus stearothermophilus/enzimologia , Listeria monocytogenes/enzimologia , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , Prótons , Teoria Quântica , Tirosina/química
9.
ACS Infect Dis ; 7(9): 2723-2735, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34432416

RESUMO

A method of creating nanoclusters (NCs) from soluble peptide molecules is described utilizing an approach based on a tyrosine-tyrosine cross-linking reaction. A reactive tag comprising histidine and tyrosine residues was introduced at the termini of the peptide molecules. The cross-linking reaction led to the creation of dityrosine bonds within the tag, which allowed for the generation of peptide NCs. We show that it is essential for the reactive tag to be present at both the "N" and "C" termini of the peptide for cluster formation to occur. Additionally, the cross-linking reaction was systematically characterized to show the importance of reaction conditions on final cluster diameter, allowing us to generate NCs of various sizes. To demonstrate the immunogenic potential of the peptide clusters, we chose to study the conserved influenza peptide, M2e, as the antigen. M2e NCs were formulated using the cross-linking reaction. We show the ability of the clusters to generate protective immunity in a dose, size, and frequency dependent manner against a lethal influenza A challenge in BALB/c mice. Taken together, the data presented suggest this new cluster formation technique can generate highly immunogenic peptide NCs in a simple and controllable manner.


Assuntos
Influenza Humana , Infecções por Orthomyxoviridae , Animais , Anticorpos Antivirais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos , Tirosina
10.
Free Radic Biol Med ; 174: 211-224, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34363946

RESUMO

An acidosis, a decrease of pH within a living tissue, may alter yields of radical reactions if participating radicals undergo partial or complete protonation. One of photosensitizers found in the human eye lens, kynurenic acid (KNA-), possesses pKa 5.5 for its radical form that is close to physiological pH 6.89 for a healthy lens. In this work we studied the influence of pH on mechanisms and products of photoinduced radical reactions between KNA- and amino acids tryptophan (Trp) and tyrosine (Tyr) within a globule of model protein, Hen White Egg Lysozyme (HEWL). Our results show that the rate constant of back electron transfer from kynurenyl to HEWL• radicals with the restoration of initial reagents - the major decay pathway for these radicals - does not change in the pH 3-7. The quantum yield of HEWL degradation is also pH independent, however a shift of pH from 7 to 5 completely changes the outcome of photoinduced damage to HEWL from intermolecular cross-linking to oxygenation. HPLC-MS analysis has shown that four of six Trp and all Tyr residues of HEWL are modified in different extents at all pH, but the lowering of pH from 7 to 5 significantly changes the direction of main photodamage from Trp62 to Trp108 located at the entrance and bottom of enzymatic center, respectively. A decrease of intermolecular cross-links via Trp62 is followed by an increase in quantities of intramolecular cross-links Tyr20-Tyr23 and Tyr23-Tyr53. The obtained results point out the competence of cross-linking and oxygenation reactions for Trp and Tyr radicals within a protein globule and significant increase of oxygenation to the total damage of protein in the case of cross-linking deceleration by coulombic repulsion of positively charged protein globules.


Assuntos
Ácido Cinurênico , Triptofano , Aminoácidos , Animais , Galinhas/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Muramidase/metabolismo , Oxirredução , Tirosina
11.
Orphanet J Rare Dis ; 16(1): 343, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344451

RESUMO

Alkaptonuria (AKU, OMIM 203500) is a rare congenital disorder caused by a deficiency of the enzyme homogentisate-1,2,-dioxygenase. The long-term consequences of AKU are joint problems, cardiac valve abnormalities and renal problems. Landmark intervention studies with nitisinone 10 mg daily, suppressing an upstream enzyme activity, demonstrated its beneficial effects in AKU patients with established complications, which usually start to develop in the fourth decade. Lower dose of nitisinone in the range of 0.2-2 mg daily will already reduce urinary homogentisic acid (uHGA) excretion by > 90%, which may prevent AKU-related complications earlier in the course of the disease while limiting the possibility of side-effects related to the increase of plasma tyrosine levels caused by nitisinone. Future preventive studies should establish the lowest possible dose for an individual patient, the best age to start treatment and also collect evidence to which level uHGA excretion should be reduced to prevent complications.


Assuntos
Alcaptonúria , Alcaptonúria/tratamento farmacológico , Cicloexanonas/uso terapêutico , Humanos , Nitrobenzoatos/uso terapêutico , Tirosina
12.
Biomolecules ; 11(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34356664

RESUMO

Aging causes oxidative stress, endothelial dysfunction and a reduction in the bioavailability of nitric oxide. The study aim was to determine whether, as a result of repeated whole-body exposure to cryogenic temperature (3 min -130 °C), there is an increase of inducible nitric oxide synthase (iNOS) concentration in senior subjects (59 ± 6 years), and if this effect is stronger in athletes. In 10 long-distance runners (RUN) and 10 untraining (UTR) men, 24 whole-body cryotherapy (WBC) procedures were performed. Prior to WBC, after 12th and 24th treatments and 7 days later, the concentration of iNOS, asymmetric dimethylarginine (ADMA), 3-nitrotyrosine (3-NTR), homocysteine (HCY), C-reactive protein (CRP) and interleukins such as: IL-6, IL-1ß, IL-10 were measured. In the RUN and UTR groups, after 24 WBC, iNOS concentration was found to be comparable and significantly higher (F = 5.95, p < 0.01) (large clinical effect size) compared to before 1st WBC and after 12th WBC sessions. There were no changes in the concentration of the remaining markers as a result of WBC (p > 0.05). As a result of applying 24 WBC treatments, using the every-other-day model, iNOS concentration increased in the group of older men, regardless of their physical activity level. Along with this increase, there were no changes in nitro-oxidative stress or inflammation marker levels.


Assuntos
Crioterapia/métodos , Óxido Nítrico Sintase Tipo II/sangue , Idoso , Arginina/análogos & derivados , Arginina/sangue , Atletas , Índice de Massa Corporal , Exercício Físico , Humanos , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Resistência Física , Tirosina/análogos & derivados , Tirosina/sangue
13.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360651

RESUMO

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.


Assuntos
Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Gases em Plasma/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo , Apoptose , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Triptofano/química , Tirosina/química
14.
Phys Chem Chem Phys ; 23(31): 16698-16706, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34338250

RESUMO

The kinetics of electron transfer (ET) from tyrosine (Tyr) to short-lived histidine (His) radicals in peptides of different structures was monitored using time-resolved chemically induced dynamic nuclear polarization (CIDNP) to follow the reduction of the His radicals using NMR detection of the diamagnetic hyperpolarized reaction products. In aqueous solution over a wide pH range, His radicals were generated in situ in the photo-induced reaction with the photosensitizer, 3,3',4,4'-tetracarboxy benzophenone. Model simulations of the CIDNP kinetics provided pH-dependent rate constants of intra- and intermolecular ET, and the pH dependencies of the reaction under study were interpreted in terms of protonation states of the reactants and the product, His with either protonated or neutral imidazole. In some cases, an increase of pKa of imidazole in the presence of the short-lived radical center at a nearby Tyr residue was revealed. Interpretation of the obtained pH dependencies made is possible to quantify the degree of paramagnetic shift of the acidity constant of the imidazole of the His residue in the peptides with a Tyr residue in its paramagnetic state, and to correlate this degree with the intramolecular ET rate constant - a higher intramolecular ET rate constant corresponded to a greater acidity constant shift.


Assuntos
Histidina/química , Peptídeos/química , Tirosina/química , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução
15.
J Biotechnol ; 339: 73-80, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34364924

RESUMO

The shape of wool yarns was changed by laccase-assisted grafting of tyrosine. Prior to tyrosine grafting a cysteine pre-treatment was optimized aiming to increase the amount of thiol reaction groups available. The best operational conditions for laccase-assisted tyrosine grafting were: i) pre-treatment with cysteine (2.2 mM) in a solution of 20 % ethanol, 15 % propylene glycol and 0.5 % benzyl alcohol, pH = 10, 40 °C; ii) tyrosine grafting with 3.0 mM tyrosine, 18 U/mL laccase, pH = 5, 40 °C. The shape modification was evaluated by number of curly twists determination on the grafted yarn samples. The thermal and mechanical properties of the grafted wool yarns was evaluated by TGA, DSC and breaking strength determination. The amount of free thiols and weight gain were assessed aiming to infer the role of the cysteine pre-treatment on the final tyrosine grafting and shape modification. The laccase-assisted grafting of tyrosine onto wool yarns have influenced the thermal and mechanical properties of the yarns however without compromising their structural integrity for the final application purposes. The developed methodology to impart new shape to wool yarns is presented herein as an environmentally friendly alternative to chemical methods. The new findings revealed great potentialities for application in similar fibers like hair.


Assuntos
Lacase , , Animais , Tirosina
16.
Molecules ; 26(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34443661

RESUMO

Protein methyltransferases are vital to the epigenetic modification of gene expression. Thus, obtaining a better understanding of and control over the regulation of these crucial proteins has significant implications for the study and treatment of numerous diseases. One ideal mechanism of protein regulation is the specific installation of a photolabile-protecting group through the use of photocaged non-canonical amino acids. Consequently, PRMT1 was caged at a key tyrosine residue with a nitrobenzyl-protected Schultz amino acid to modulate protein function. Subsequent irradiation with UV light removes the caging group and restores normal methyltransferase activity, facilitating the spatial and temporal control of PRMT1 activity. Ultimately, this caged PRMT1 affords the ability to better understand the protein's mechanism of action and potentially regulate the epigenetic impacts of this vital protein.


Assuntos
Epigênese Genética/efeitos da radiação , Proteínas Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos/genética , Aminoácidos , Epigênese Genética/genética , Expressão Gênica/efeitos da radiação , Humanos , Metilação/efeitos da radiação , Proteínas Metiltransferases/efeitos da radiação , Proteína-Arginina N-Metiltransferases/efeitos da radiação , Proteínas Repressoras/efeitos da radiação , Fatores de Transcrição/genética , Tirosina/química , Raios Ultravioleta
17.
Biomolecules ; 11(7)2021 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-34356675

RESUMO

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Epigênese Genética , Humanos , Lisina/química , Lisina/metabolismo , Simulação de Dinâmica Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerização Proteica , Prótons , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tirosina/química , Tirosina/metabolismo
18.
ACS Chem Neurosci ; 12(17): 3237-3249, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34406754

RESUMO

There is a plethora of significant research that illustrates toxic self-assemblies formed by the aggregation of single amino acids, such as phenylalanine, tyrosine, tryptophan, cysteine, and methionine, and their implication on the etiology of inborn errors of metabolisms (IEMs), such as phenylketonuria, tyrosinemia, hypertryptophanemia, cystinuria, and hypermethioninemia, respectively. Hence, studying the aggregation behavior of single amino acids is very crucial from the chemical neuroscience perspective to understanding the common etiology between single amino acid metabolite disorders and amyloid diseases like Alzheimer's and Parkinson's. Herein we report the aggregation properties of nonaromatic single amino acids l-proline (Pro), l-hydroxyproline (Hyp), and l-lysine hydrochloride (Lys). The morphologies of the self-assembled structures formed by Pro, Hyp, and Lys were extensively studied by various microscopic techniques, and controlled morphological transitions were observed under varied concentrations and aging times. The mechanism of structure formation was deciphered by concentration-dependent 1H NMR analysis, which revealed the crucial role of hydrogen bonding and hydrophobic interactions in the structure formation of Pro, Hyp, and Lys. MTT assays on neural (SHSY5Y) cell lines revealed that aggregates formed by Pro, Hyp, and Lys reduced cell viability in a dose-dependent manner. These results may have important implications in the understanding of the patho-physiology of disorders such as hyperprolinemia, hyperhydroxyprolinemia, and hyperlysinemia since all these IEMs are associated with severe neurodegenerative symptoms, including intellectual disability, seizures, and psychiatric problems. Our future studies will endeavor to study these biomolecular assemblies in greater detail by immuno-histochemical analysis and advanced biophysical assays.


Assuntos
Lisina , Prolina , Aminoácidos , Hidroxiprolina , Tirosina
19.
Adv Exp Med Biol ; 1288: 1-20, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34453729

RESUMO

Non-receptor tyrosine kinases (NRTKs) are implicated in various biological processes including cell proliferation, differentiation, survival, and apoptosis, as well as cell adhesion and movement. NRTKs are expressed in all mammals and in different cell types, with extraordinarily high expression in the testis. Their association with the plasma membrane and dynamic subcellular localization are crucial parameters in their activation and function. Many NRTKs are found in endosomal protein trafficking pathways, which suggests a novel mechanism to regulate the timely junction restructuring in the mammalian testis to facilitate spermiation and germ cell transport across the seminiferous epithelium.


Assuntos
Epitélio Seminífero , Espermatogênese , Animais , Adesão Celular , Masculino , Células de Sertoli , Testículo , Tirosina
20.
Anal Chem ; 93(29): 10334-10342, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34251175

RESUMO

The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, two different probes were evaluated, and the probe with the best performance was employed to further study the tyrosine residues in the human proteome. Then, tagged tyrosine-containing peptides were selectively enriched using bioorthogonal chemistry, and after the cleavage, a small tag on the peptides perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5000 tyrosine sites in MCF7 cells, and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.


Assuntos
Proteoma , Tirosina , Humanos , Ligantes , Ligação Proteica , Proteômica
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