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1.
Biomater Sci ; 9(6): 1961-1973, 2021 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-33537687

RESUMO

Methionine (Met), an essential amino acid in the human body, possesses versatile features based on its chemical modification, cell metabolism and metabolic derivatives. Benefitting from its multifunctional properties, Met holds immense potential for biomedical applications. In this review, we systematically summarize the recent progress in Met-based strategies for biomedical applications. First, given the unique structural characteristics of Met, two chemical modification methods are briefly introduced. Subsequently, due to the disordered metabolic state of tumor cells, applications of Met in cancer treatment and diagnosis are summarized in detail. Furthermore, the efficacy of S-adenosylmethionine (SAM), as the most important metabolic derivative of Met, for treating liver diseases is mentioned. Finally, we analyze the current challenges and development trends of Met in the biomedical field, and suggest that Met-restriction therapy might be a promising approach to treat COVID-19.


Assuntos
Metionina/metabolismo , Neoplasias/metabolismo , /tratamento farmacológico , /virologia , Proliferação de Células/efeitos dos fármacos , Docetaxel/química , Docetaxel/farmacologia , Humanos , Hepatopatias/dietoterapia , Hepatopatias/patologia , Metionina/química , Metionina/deficiência , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , S-Adenosilmetionina/uso terapêutico , /isolamento & purificação
2.
Nat Commun ; 12(1): 1086, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597529

RESUMO

The dynamics of photodissociation and recombination in heme proteins represent an archetypical photochemical reaction widely used to understand the interplay between chemical dynamics and reaction environment. We report a study of the photodissociation mechanism for the Fe(II)-S bond between the heme iron and methionine sulfur of ferrous cytochrome c. This bond dissociation is an essential step in the conversion of cytochrome c from an electron transfer protein to a peroxidase enzyme. We use ultrafast X-ray solution scattering to follow the dynamics of Fe(II)-S bond dissociation and 1s3p (Kß) X-ray emission spectroscopy to follow the dynamics of the iron charge and spin multiplicity during bond dissociation. From these measurements, we conclude that the formation of a triplet metal-centered excited state with anti-bonding Fe(II)-S interactions triggers the bond dissociation and precedes the formation of the metastable Fe high-spin quintet state.


Assuntos
Citocromos c/metabolismo , Compostos Ferrosos/metabolismo , Ferro/metabolismo , Metais/metabolismo , Metionina/metabolismo , Citocromos c/química , Transporte de Elétrons/efeitos da radiação , Compostos Ferrosos/química , Heme/química , Heme/metabolismo , Ferro/química , Metais/química , Metionina/química , Simulação de Dinâmica Molecular , Fotólise , Espectrometria por Raios X
3.
Jpn J Clin Oncol ; 50(5): 512-518, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32129443

RESUMO

BACKGROUND: Primary central nervous system lymphoma (PCNSL) responds relatively quickly to chemotherapy or radiotherapy. However, determination of a complete response after treatment is often difficult because of extremely light residual contrast enhancement on magnetic resonance images due to the effects of microhemorrhages and scar tissue formation. These small enhancing lesions define an unconfirmed complete response. The aim of this study was to investigate the usefulness of carbon-11-labeled methionine (11C-Met) positron-emission tomography (PET) for determining the treatment response of PCNSL. METHODS: Data for 36 patients who were treated for PCNSL between 2011 and 2015 and underwent magnetic resonance imaging and 11C-Met PET were reviewed. Magnetic resonance imaging findings were classified as complete response, unconfirmed complete response, and tumor mass (a composite of partial response, stable disease and progressive disease). PET images were evaluated, standardized uptake values were quantified, and the tumor-to-normal tissue count ratio (TNR) was calculated. Receiver operating characteristic curves were generated to determine the optimal cutoff TNRs. RESULTS: The optimal TNRs for differentiating complete response and unconfirmed complete response from tumor mass were 1.83 (area under the curve, 0.951) and 1.80 (area under the curve, 0.932), respectively. The corresponding sensitivity and specificity values for the diagnosis of tumor mass were 82.4 and 100%, respectively, in the complete response group and 85.3 and 85%, respectively, in the unconfirmed complete response group. CONCLUSIONS: A TNR of ≥1.80 can aid in the detection of active PCNSL using 11C-Met PET. Thus, 11C-Met-PET may be a useful tool for accurate evaluation of the treatment efficacy in PCNSL.


Assuntos
Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Neoplasias do Sistema Nervoso Central/terapia , Metionina/química , Tomografia por Emissão de Pósitrons , Adulto , Idoso , Neoplasias do Sistema Nervoso Central/diagnóstico , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Curva ROC , Resultado do Tratamento
4.
Chem Commun (Camb) ; 56(26): 3741-3744, 2020 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-32124910

RESUMO

Continuous efforts have been invested in the selective modification of proteins. Herein, we first report the construction of sulfonium tethered cyclic peptides via an intramolecular cyclization by an aliphatic halide. This cyclization could enhance the stability and cellular uptake of peptides. Furthermore, the sulfonium center could be recognized by cysteine in the vicinity of the protein-peptide interacting interface and form a peptide-protein conjugate.


Assuntos
Metionina/química , Peptídeos Cíclicos/química , Compostos de Sulfônio/química , Alquilação , Transporte Biológico , Ciclização , Células HeLa , Humanos , Peptídeos Cíclicos/farmacologia
5.
Eur J Pharm Biopharm ; 149: 1-11, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32006605

RESUMO

We have observed an interesting phenomenon in which grinding of freeze-dried monoclonal antibody X (mAb-X) formulation powder resulted to significant protein sub-visible particles (SbVPs) in the reconstituted liquid, which could only be observed by sensitive particle analytical methods such as MFI and DLS. Effects of grinding temperature and the free radical scavengers methionine and 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidin-yloxy free radical (CTPO) on the formation of SbVPs were also evaluated. Free radicals were observed by EPR and the amount of free radicals was correlated to the sample temperature prior to grinding. Formation of SbVPs could be partially inhibited by methionine and CTPO. The amount of SbVPs formed was dependent on the amount of free radicals/sample temperature prior to grinding. At higher temperatures, more free radicals and SbVPs formed. Other than the previously known protein degradation due to high temperature formed during mechanical grinding, we propose an unreported and supplementary mechanism, i.e., the formation of free radicals (i.e., due to break of CO or CS bonds) in the dried state during mechanical grinding, leading to protein particle formation in the reconstituted solution. Our observation suggested that mechanical grinding of protein powder should be avoided or used cautiously (i.e., grinding temperature, strength and time) and the effects on radical and particle formation be fully evaluated.


Assuntos
Anticorpos Monoclonais/química , Química Farmacêutica , Radicais Livres/química , Proteínas/química , Óxidos N-Cíclicos/química , Depuradores de Radicais Livres/química , Liofilização , Metionina/química , Pós , Temperatura
6.
Life Sci ; 253: 117360, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32001269

RESUMO

AIMS: Progesterone receptor membrane component 1 (PGRMC1) has been reported to mediate the neuroprotective effect of progesterone, but the exact mechanism has not been elucidated. Therefore, the purpose of this study was to investigate the signalling pathway downstream of PGRMC1 in progesterone-induced neuroprotection. Recognition of the mechanism of progesterone opens novel perspectives for the treatment of diseases of the nervous system. MAIN METHODS: The PGRMC1 protein level was knocked down in rat primary cortical neurons, and Aß25-35 was used to establish an Alzheimer's disease cell model. The neuroprotective effect of progesterone was assessed by Hoechst 33258 staining and a cell counting kit-8 (CCK-8) assay. Then, proteomic and bioinformatic methods were used to analyse the proteins altered in response to PGRMC1 silencing to identify target proteins and signalling pathways involved in PGRMC1-mediated progesterone-induced neuroprotection. These findings were further verified by using signalling pathway inhibitors and western blotting. KEY FINDINGS: The neuroprotective effect of progesterone was significantly attenuated with PGRMC1 silencing. The expression of many proteins in the Ras signalling pathway was significantly changed in response to PGRMC1 silencing. FTI-277 inhibited progesterone-induced neuroprotection. Progesterone increased the expression of total Ras and Grb2. SIGNIFICANCE: These findings provide new perspectives for understanding the mechanism of and role of PGRMC1 in progesterone-induced neuroprotection. The Ras signalling pathway is the signalling pathway downstream of PGRMC1 in the mediation of progesterone-induced neuroprotection.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Proteínas de Membrana/metabolismo , Neuroproteção/efeitos dos fármacos , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Apoptose , Sobrevivência Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2/metabolismo , Técnicas de Inativação de Genes/métodos , Inativação Gênica , Humanos , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteômica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Espectrometria de Massas em Tandem
7.
Chem Commun (Camb) ; 56(21): 3175-3178, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32065188

RESUMO

We present an autocatalytic system for the detection and amplification of thiols termed the Methionase Chain Reaction (MCR). MCR is based on the reversible modification of the thiol producing enzyme Methionine Gamma-Lyase (MGL). MCR was able to amplify the concentration of thiols by a factor of 560 and was able to visually detect thiols at concentrations as low as 50 nM.


Assuntos
Liases de Carbono-Enxofre/química , Compostos de Sulfidrila/análise , Catálise , Colorimetria , Corantes/química , Limite de Detecção , Metionina/química , Oxirredução
8.
J Phys Chem Lett ; 11(4): 1215-1221, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31978303

RESUMO

Site-selective dissociation induced by core photoexcitation of biomolecules is of key importance for the understanding of radiation damage processes and dynamics and for its promising use as "chemical scissors" in various applications. However, identifying products of site-selective dissociation in large molecules is challenging at the carbon, nitrogen, and oxygen edges because of the high recurrence of these atoms and related chemical groups. In this paper, we present the observation of site-selective dissociation at the sulfur L-edge in the gas-phase peptide methionine enkephalin, which contains only a single sulfur atom. Near-edge X-ray absorption mass spectrometry has revealed that the resonant S 2p → σ*C-S excitation of the sulfur contained in the methionine side chain leads to site-selective dissociation, which is not the case after core ionization above the sulfur L-edge. The prospects of such results for the study of charge dynamics in biomolecular systems are discussed.


Assuntos
Gases/química , Peptídeos/química , Enxofre/química , Espectroscopia por Absorção de Raios X , Encefalinas/química , Metionina/química , Prótons , Teoria Quântica
9.
Sci Rep ; 10(1): 268, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937809

RESUMO

Oxidative stress has been linked to a number of chronic diseases, and this has aroused interest in the identification of clinical biomarkers that can accurately assess its severity. We used liquid chromatography-high resolution mass spectrometry (LC-MS) to show that oxidised and non-oxidised Met residues at position 147 of human serum albumin (Met147) can be accurately and reproducibly quantified with stable isotope-labelled peptides. Met147 oxidation was significantly higher in patients with diabetes than in controls. Least square multivariate analysis revealed that glycated haemoglobin (HbA1c) and glycated albumin (GA) did not significantly influence Met147 oxidation, but the GA/HbA1c ratio, which reflects glycaemic excursions, independently affected Met147 oxidation status. Continuous glucose monitoring revealed that Met147 oxidation strongly correlates with the standard deviation of sensor glucose concentrations and the time spent with hypoglycaemia or hyperglycaemia each day. Thus, glycaemic variability and hypoglycaemia in diabetes may be associated with greater oxidation of Met147. Renal function, high-density lipoprotein-cholesterol and serum bilirubin were also associated with the oxidation status of Met147. In conclusion, the quantification of oxidised and non-oxidised Met147 in serum albumin using our LC-MS methodology could be used to assess the degree of intravascular oxidative stress induced by hypoglycaemia and glycaemic fluctuations in diabetes.


Assuntos
Biomarcadores/sangue , Complicações do Diabetes/patologia , Hipoglicemia/patologia , Metionina/química , Albumina Sérica Humana/análise , Idoso , Bilirrubina/sangue , Glicemia/análise , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Hemoglobina A Glicada/análise , Humanos , Hipoglicemia/complicações , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Oxirredução , Estresse Oxidativo , Peptídeos/análise , Albumina Sérica/análise , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo
10.
PLoS Comput Biol ; 16(1): e1007600, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917825

RESUMO

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.


Assuntos
Enzimas , Método de Monte Carlo , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Especificidade por Substrato/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Azidas/química , Azidas/metabolismo , Sítios de Ligação/genética , Catálise , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Metionina tRNA Ligase/química , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Mutação/genética , Norleucina/análogos & derivados , Norleucina/química , Norleucina/metabolismo
11.
J Inorg Biochem ; 203: 110885, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31731049

RESUMO

The synthesized 2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenanthroline (HNAIP) ligand and its new iridium ([Ir(ppy)2(HNAIP)]Cl) and rhodium ([Rh(ppy)2(HNAIP)]Cl) complexes, being ppy = 2-phenylpiridinate, show cytotoxic effects in SW480 (colon adenocarcinoma) and A549 (epithelial lung adenocarcinoma) cells. They all are cytotoxic in the tested cell lines. HNAIP and [Rh(ppy)2(HNAIP)]+ are the most cytotoxic, whereas [Ir(ppy)2(HNAIP)]+ displays negligible cytotoxicity towards A549 cells and moderate activity towards SW480. The interaction of all three compounds with Bovine Serum Albumin (BSA), l-glutathione reduced (GSH), nicotinamide adenine dinucleotide (NADH) and DNA was studied to explain the differences found in terms of cytotoxicity. None of them are able to interact with BSA, thus excluding bioavailability due to plasma protein interaction as the possible differentiating factor in their biological activity. By contrast, small differences have been observed regarding DNA interaction. In addition, taking advantage of the emission properties of these molecules, they have been visualized in the cytoplasmic region of A549 cells. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show, in turn, that the internalization ability follow the sequence [Rh(ppy)2(HNAIP)]+ > [Ir(ppy)2(HNAIP)]+ > cisplatin. Therefore, it seems clear that the cellular uptake by tumour cells is the key factor affecting the different cytotoxicity of the metal complexes and that this cellular uptake is influenced by the hydrophobicity of the studied complexes. On the other hand, preliminary catalytic experiments performed on the photo-oxidation of GSH and some amino acids such as l-methionine (Met), l-cysteine (Cys) and l-tryptophan (Trp) provide evidence for the photocatalytic activity of the Ir(III) complex in this type of reactions.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Fenantrolinas/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/efeitos da radiação , Catálise , Bovinos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , Complexos de Coordenação/efeitos da radiação , Cisteína/química , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa/química , Humanos , Irídio/química , Irídio/efeitos da radiação , Ligantes , Luz , Metionina/química , Oxirredução , Fenantrolinas/síntese química , Fenantrolinas/metabolismo , Fenantrolinas/efeitos da radiação , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/efeitos da radiação , Ródio/química , Ródio/efeitos da radiação , Triptofano/química
12.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140334, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31786473

RESUMO

Aseptic processing and terminal sterilization become increasingly challenging as medical devices become more complex and include active biologics. Terminal sterilization is preferred for patient safety and production costs. We aimed to determine how sterilization using supercritical CO2 (scCO2) with low levels of peracetic acid (PAA) affects amino acids and human epidermal growth factor (EGF) as a model protein. In a benchtop reactivity test, the amino acids methionine, tryptophan, arginine and lysine reacted with low levels of PAA in solution. At PAA levels used for scCO2 sterilization, however, mass spectrometry only identified oxidative adducts on methionine and tryptophan. Mass spectrometry analysis of EGF exposed to scCO2/PAA identified oxidative adducts on residues Met21, Trp49 and Trp50, as well as a low level of truncations after residues Trp49 and Trp50. Importantly, processing of EGF in solution with scCO2 did not affect its native conformation, and sterilized EGF maintained its activity in cell proliferation assays. When processing samples in lyophilized form with scCO2/PAA, amino acids did not react with PAA and the presence of adducts was strongly reduced on methionine and tryptophan, both as single amino acids and in EGF. Truncation after tryptophan residues did not occur. EGF sterilized in the lyophilized form retained its activity when processing occurred with added moisture. These results have significant implications for the maintenance of biological function in sterilized decellularized scaffolds and the ability to manufacture terminally sterilized combination devices containing therapeutic peptides or proteins.


Assuntos
Dióxido de Carbono/química , Fator de Crescimento Epidérmico/química , Ácido Peracético/química , Esterilização , Células 3T3 , Animais , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Humanos , Espectrometria de Massas , Metionina/análise , Metionina/química , Camundongos , Oxirredução , Triptofano/análise , Triptofano/química
13.
Chemistry ; 26(1): 259-268, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31614021

RESUMO

In the effort to overcome issues of toxicity and resistance inherent to treatment by the approved platinum anticancer agents, a large number of cisplatin variants continues today to be prepared and tested. One of the applied strategies is to use monofunctional platinum complexes that, unlike traditional bifunctional compounds, are able to form only a single covalent bond with nuclear DNA. Chirality, aquation reaction, interaction with guanine and N-acetyl methionine as well as, intercalation into, binding to and distortion of DNA have been investigated by using both quantum mechanical DFT and molecular dynamics computations aiming at contributing to the elucidation of the molecular mechanism underlying the significantly enhanced spectrum of activity of the monofunctional PtII drug phenanthriplatin. Analogous calculations have been performed in parallel for other two less potent monofunctional PtII drugs, pyriplatin and enpyriplatin, which show very different cytotoxic effects.


Assuntos
Antineoplásicos/química , Teoria da Densidade Funcional , Simulação de Dinâmica Molecular , Compostos Organoplatínicos/química , Fenantridinas/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Humanos , Substâncias Intercalantes/química , Metionina/química , Conformação de Ácido Nucleico , Compostos Organoplatínicos/farmacologia , Fenantridinas/farmacologia , Termodinâmica , Transcrição Genética/efeitos dos fármacos
14.
Biochemistry ; 59(2): 132-138, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31592657

RESUMO

Methionine is one of the most hydrophobic, redox-sensitive, and one of the only two sulfur-containing amino acids on protein. Because of these biochemical properties, the methionine residue plays a central role in a variety of biological processes, such as metal coordination, antioxidant stress, and aging. However, studies on the molecular functions of methionine are much less common than the other primary sulfur-containing amino acid, cysteine. The limited number of publications on methionine-related studies is partially due to the lack of tools for methionine modification. Methionine bioconjugation offers a new strategy to decipher the biological function of methionine and expands the toolbox for protein functionalization in the context of the application, such as synthesizing proteins with novel properties and producing new biomaterials. The purpose of this Perspective is to highlight the biochemical properties and functions of methionine, list recent progress in the development of methionine bioconjugation reagents, and briefly demonstrate the application of these reagents on polypeptides, proteins, and proteomes.


Assuntos
Metionina/química , Peptídeos/química , Proteínas/química , Proteoma/química , Alquilação , Indicadores e Reagentes/química , Oxirredução
15.
Biochim Biophys Acta Gen Subj ; 1864(1): 129453, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676294

RESUMO

BACKGROUND: L-Homocysteine (Hcy) is a non-proteinogenic α-amino acid synthesized from dietary methionine. In healthy humans, high Hcy levels are a risk factor for cardiovascular diseases, stroke and type 2 diabetes. A recent study reports that Hcy reacts with Cys10 of transthyretin (TTR), generating a stable covalent adduct. However, to date the effect of S-homocysteinylation on TTR conformational stability remains unknown. METHODS: The effect of Hcy on the conformational properties of wt- and L55P-TTR were analysed using a set of biophysical techniques. The cytotoxicity of S-homocysteinylated L55P-TTR was also evaluated in the HL-1 cardiomyocyte cell line, while the effects of the assemblies on kinematic and dynamics properties of cardiac muscle cells were analysed in cardiomyocyte syncytia. RESULTS: We found that Hcy stabilizes tetrameric wt-TTR, while it destabilizes the tetrameric structure of the L55P mutant, promoting the accumulation of self-assembly-prone monomeric species. CONCLUSIONS: Our study demonstrated that S-homocysteinylation of the L55P-TTR mutant impairs protein stability, favouring the appearance of toxic monomers. Interestingly, S-homocysteinylation affected only mutant, not wt-TTR. Moreover, we also show that assemblies of S-homocysteinylated L55P-TTR impair cardiomyocytes functional parameters. GENERAL SIGNIFICANCE: Our study offers new insights on the negative impact of S-homocysteinylation on L55P-TTR stability, whose aggregation is considered the causative agent of a form of early-onset familial amyloid polyneuropathy and cardiomyopathy. Our results suggest that high homocysteine levels are a further risk factor for TTR cardiomyopathy in patients harbouring the L55P-TTR mutation.


Assuntos
Neuropatias Amiloides Familiares/genética , Cardiomiopatias/genética , Homocisteína/genética , Pré-Albumina/química , Neuropatias Amiloides Familiares/patologia , Cardiomiopatias/patologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Homocisteína/química , Humanos , Metionina/química , Mutação/genética , Miócitos Cardíacos , Pré-Albumina/genética , Pré-Albumina/ultraestrutura , Conformação Proteica , Estabilidade Proteica , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Relação Estrutura-Atividade
16.
Phys Chem Chem Phys ; 22(1): 107-113, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31807746

RESUMO

The non-enzymatic cleavage rates of amide bonds located in peptides in aqueous solution is pH-dependent and involves two distinct mechanisms: direct hydrolysis (herein termed "scission") and intramolecular aminolysis by the N-terminal amine (herein termed "backbiting"). While amide bond cleavage has been previously characterized using a variety of peptides, no systematic study has yet been reported addressing the effect of the pH on the interplay between the two amide bond cleavage pathways. In this study, the cleavage rates of the glycine dimer (GG), the glycine trimer (GGG), and the cyclic dimer (cGG), as well as the alanine trimer (AAA), were measured at pH 3, 5, 7, and 10 at 95 °C employing quantification based on 1H NMR. The distinct rate constants for scission and backbiting processes were obtained by solving the differential rate equations associated with the proposed kinetic model. Generalizations concerning the relative importance of the various amide bond cleavage pathways at pH 3, 5, 7, and 10 are presented. In particular, scission dominates at pH 10, while backbiting dominates at neutral pH. At the acidic pH of 3, both backbiting and scission are significant. The model of the reaction network, used in this work, enables the quantification of these multiple competing mechanisms and can be applied to longer peptides and to similar types of reaction networks.


Assuntos
Concentração de Íons de Hidrogênio , Peptídeos/química , Alanina/química , Amidas/química , Aminas/química , Glicina/química , Hidrólise , Cinética , Metionina/química , Estabilidade Proteica , Termodinâmica
17.
Food Res Int ; 126: 108580, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732086

RESUMO

During storage and processing of foods, myofibrillar proteins (MP), the most abundant proteins of meats, are exposed to peroxyl radicals (ROO). The present work shows that ROO induce oxidation of MP leading to a widespread of MP aggregation. In spite of the extent of such process, only partial consumption of the more oxidizable amino acids was determined. MP were exposed to ROO derived from thermolysis of AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride), and samples studied through SDS-PAGE, western blotting, light scattering, time-resolved fluorescence, and high performance liquid chromatography. Together with MP aggregation, consumption of methionine (the most consumed residue), cysteine, tyrosine, and tryptophan were determined. These results are associated with conformational changes of MP affecting the accessibility of tryptophan residues to the solvent, as evidenced by a decreasing of its fluorescence lifetime. Lysine residues, which are not reactive towards ROO, were also consumed, suggesting participation of Schiff bases in the MP aggregation process.


Assuntos
Aminoácidos/química , Proteínas de Transporte/química , Peróxidos/química , Cisteína/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Carne/química , Metionina/química , Oxirredução , Triptofano/química , Tirosina/química
18.
Proc Natl Acad Sci U S A ; 116(48): 24359-24365, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719194

RESUMO

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.


Assuntos
Metionina/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cloraminas/química , Escherichia coli/genética , Temperatura Alta , Humanos , Peróxido de Hidrogênio/química , Macrófagos , Metionina/química , Mutação , Oxidantes/química , Oxirredução , Técnicas de Patch-Clamp , Fagocitose , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Compostos de Tosil/química
19.
J Anim Sci ; 97(12): 4746-4760, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31679027

RESUMO

This study examined the influence of a diet enriched with free methionine (dl-Met) or methionine dipeptide (dl-MMet) on the intestinal health of Eimeria-challenged (EC) and unchallenged (UC) broilers. A non-supplemented, methionine-deficient diet (NS) was used as control. Treatments were arranged in a 2 × 3 factorial completely randomized design with eight replications. Broilers in the EC group were infected with sporulated oocysts of Eimeria spp. (E. acervulina, E. maxima, E. praecox, and E. mitis) at 14 d of age. Performance analysis, light and electron microscopy of the jejunum, analysis of genes related to apoptosis and cell proliferation in the jejunum, and blood tests were performed at 6 days post-inoculation (dpi). EC broilers had poorer performance than UC broilers, regardless of diet (P < 0.001). Broilers fed the dl-Met diet had greater weight gain (P = 0.004) and lower feed conversion ratio (P = 0.019) than broilers fed other diets. Jejunal sections from EC broilers fed the NS diet showed short (P = 0.001) and wide villi (P < 0.001) with increased crypt depth (P < 0.001) and reduced villus / crypt ratio (P = 0.001), jejunal absorptive surface area (P < 0.001), number of neutral goblet cells (Eimeria challenge: P = 0.048; diet P = 0.016), and mucin 2 (MUC2) gene expression (P = 0.018). EC birds fed the dl-MMet diet had higher enterocyte height (P < 0.001). Birds fed the dl-MMet diet had low lamina propria width (P = 0.009). UC broilers fed the dl-Met diet had the highest number of acidic goblet cells (P = 0.005), whereas EC broilers assigned the dl-MMet diet showed the highest number of intraepithelial lymphocytes (P = 0.033). Reduced expression of caspase-3 (CASP3) (P = 0.005), B-cell lymphoma 2 (BCL2) (P < 0.001), mechanistic target of rapamycin (MTOR) (P < 0.001), and ribosomal protein S6 kinase B1 (RPS6KB1) (P < 0.001) genes was observed in EC animals. MTOR expression levels were highest in birds fed the dl-MMet diet (P = 0.004). Plasma activities of aspartate aminotransferase (AST) was influenced by both diet (P = 0.002) and Eimeria challenge (P = 0.005), with EC broilers assigned the NS diet showing the highest levels. EC broilers fed the NS diet had higher creatine kinase (CK) activity (P = 0.049). EC broilers had lower plasma uric acid (P = 0.004) and higher serum mucoproteins level (P < 0.001). These results indicate that methionine dipeptide supplementation is able to mitigate the harmful intestinal effects of Eimeria spp. in broilers.


Assuntos
Coccidiose/veterinária , Metionina/farmacologia , Doenças das Aves Domésticas/prevenção & controle , Ração Animal/análise , Animais , Galinhas , Coccidiose/prevenção & controle , Dieta/veterinária , Suplementos Nutricionais/análise , Eimeria , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , Metionina/administração & dosagem , Metionina/química , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/parasitologia
20.
J Colloid Interface Sci ; 557: 777-792, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31580974

RESUMO

Positively charged elastin-like polypeptides (ELPs) were synthesized for the compaction of genetic material. A recombinant ELP (VPGXG)40 with X = V,M (3:1) was post-modified in two steps to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. Positively charged ELPs were characterized by SDS-PAGE, size exclusion chromatography, 1H NMR, potentiometric titrations and dynamic light scattering to assess their purity and determine their degree of functionalization, molecular weight, isoelectric point and thermo-responsive behaviour. Electrostatic complexation between the different ELP derivatives and nucleic acids was studied to determine the stoichiometry of ELPS/nucleic acids complex formation, and to find optimal conditions leading to stable nanoparticles with controlled size and surface potential. The stability of these complexes was investigated in the presence of salts at physiological concentrations and in the presence of surfactant. This study revealed that two regimes of stable nanoparticles in terms of size and charge can be obtained from the electrostatic complexation between the primary amine containing ELP derivative, ELP(-NH2), and plasmid DNA. Resulting complexes were found to be stable to dissociation for charge ratios up to 2.5 under physiological salt concentrations (154 mM NaCl), showing that plasmid DNA was completely condensed by the polycationic ELP and protected against electrolyte-mediated dissociation.


Assuntos
Elastina/química , Nanopartículas/química , Ácidos Nucleicos/química , Peptídeos/química , Alquilação , Aminas/química , Cátions/química , Interações Hidrofóbicas e Hidrofílicas , Metionina/química , Conformação Molecular , Peso Molecular , Polieletrólitos/química , Multimerização Proteica , Eletricidade Estática
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