RESUMO
Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.
Assuntos
Arginase/ultraestrutura , Arginina/genética , Ácido Aspártico/genética , Conformação Proteica , Arginase/química , Arginase/genética , Arginina/química , Ácido Aspártico/química , Ácido Glutâmico/química , Ácido Glutâmico/genética , Humanos , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Mutação/genética , Multimerização Proteica/genéticaRESUMO
The aggregation of proteins in the brain is one of the main features of neurodegenerative diseases. In Alzheimer's disease, the abnormal aggregation of Aß-42 is due to intrinsic and extrinsic factors. The latter is due to variations in the environment, such as temperature, salt concentration, and pH. We evaluated the effect of protonation/deprotonation of residues that are part of trimeric and pentameric oligomers at pH 5, pH 6, and pH 7. Molecular dynamics simulation at 200 ns in the canonical ensemble was implemented. The results have revealed that histidine, glutamic acid, and aspartic acid residues showed a protonation/deprotonation effect in oligomers. The root mean square deviation analysis was used to analyze the structural stability at different pHs. We found an increase in hydrophobicity in the side chains of the trimer, while in the pentamer, the structural instability of a compact structure at pH 5 caused the hydrophobic core to open, revealing the hydrophobic region to the environment. At this point, we believe that conformational changes mediated by pH are essential in the aggregation of Aß-42 oligomers.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/genética , Doença de Alzheimer/patologia , Sequência de Aminoácidos/genética , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Ácido Aspártico/genética , Histidina/química , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Multimerização Proteica/genéticaRESUMO
We aimed to evaluate whether variants in repair (XPD Asp312Asn, XPD Lys751Gln) and detoxification (GSTM1, GSTT1) genes alter risk, clinicopathological aspects and survival of cutaneous melanoma (CM). Genotyping was performed in 229 CM patients and 258 controls. Individuals with XPD 312Asp/Asn or Asn/Asn plus GSTT1 null genotype were under 2.00 (95% CI: 1.06-3.79), and XPD 312Asn/Gln haplotype was under 1.44-fold (95% CI: 0.99-2.08) increased risks to CM than others. Individuals with GSTM1 plus GSTT1 null genotype had 9.61-fold (95% CI: 2.28-40.38) increased risk of metastatic CM. At 60 months of follow-up, patients with XPD 751Gln/Gln plus GSTT1 null and GSTM1 null plus GSTT1 null genotype presented 7.36 and 3.05 more chances of evolving to death in multivariate Cox analysis, respectively. In conclusion, our data indicate, for the first time, that specific variant combinations of XPD, GSTM1 and GSTT1 may increase susceptibility to CM and influence patients' clinicopathological features and survival.
Assuntos
Glutationa Transferase/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Xeroderma Pigmentoso/diagnóstico , Xeroderma Pigmentoso/genética , Idoso , Asparagina/genética , Ácido Aspártico/genética , Sobrevivência Celular , Intervalo Livre de Doença , Predisposição Genética para Doença , Variação Genética , Genótipo , Glutamina/genética , Humanos , Lisina/genética , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Resultado do TratamentoRESUMO
NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using 1 H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.
Assuntos
Histona Acetiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Metaboloma/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alanina/genética , Ácido Aspártico/genética , Regulação da Expressão Gênica/genética , Humanos , Metabolômica/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Prolina/genética , Domínios Proteicos/genética , Transdução de Sinais/genéticaAssuntos
Humanos , Adulto , Tirosina/genética , Ácido Aspártico/genética , Hepatite B Crônica/genética , Metionina/genética , Mutação/genética , Tirosina/uso terapêutico , China , Lamivudina/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Motivos de Aminoácidos/genética , Reação em Cadeia da Polimerase em Tempo RealAssuntos
Ácido Aspártico/genética , Hepatite B Crônica/genética , Metionina/genética , Mutação/genética , Tirosina/genética , Adulto , Motivos de Aminoácidos/genética , Fármacos Anti-HIV/uso terapêutico , China , Hepatite B Crônica/tratamento farmacológico , Humanos , Lamivudina/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Tirosina/uso terapêuticoRESUMO
BACKGROUND: Polymorphisms in the endothelial nitric oxide synthase (eNOS) and in the plasminogen activator inhibitor -1 (PAI-1) genes have been implicated in stroke pathogenesis but results are still controversial. The aim of this study was to examine the possible contribution of Glu298Asp in the eNOS and 4G/5G in the PAI-1polymorphisms with ischemic stroke in a young Mexican population. MATERIALS AND METHODS: In a case-control study, conducted between January 2006 and June 2010, 204 patients ≤45 years of age with ischemic stroke and 204 controls matched by age and gender, were recruited. The Glu298Asp and 4G/5G polymorphisms were determined in all participants by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: There was a significant difference in the Glu298Asp genotype distribution (P=0.001) and allele frequency between the two groups (P=0.001). The 4G/5G genotype distribution (P=0.40) and the allele frequency was similar between groups; (P=0.13). There were independent factors for ischemic stroke: Asp carriage (GluAsp+AspAsp) (P=0.02); smoking (P=0.01); hypertension (P=0.03), and familial history of atherothrombotic disease (P=0.04). CONCLUSIONS: The Asp allele from the Gu298Asp gene represents an independent risk factor for ischemic stroke in a young Mexican population. In contrast, the 4G/5G was not associated with an increased risk for this disease in the same group of patients, as previously has been demonstrated in other populations.
Assuntos
Predisposição Genética para Doença/genética , Óxido Nítrico Sintase Tipo III/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Polimorfismo Genético/genética , Acidente Vascular Cerebral/genética , Adulto , Ácido Aspártico/genética , Isquemia Encefálica/complicações , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Ácido Glutâmico/genética , Humanos , Masculino , México , Acidente Vascular Cerebral/etiologiaRESUMO
The water cavity of yeast thioredoxin 1 (yTrx1) is an ancestral, conserved structural element that is poorly understood. We recently demonstrated that the water cavity is involved in the complex protein dynamics that are responsible for the catalytically relevant event of coupling hydration, proton exchange, and motion at the interacting loops. Its main feature is the presence of the conserved polar residue, Asp24, which is buried in a hydrophobic cavity. Here, we evaluated the role of the solvation of Asp24 as the main element that is responsible for the formation of the water cavity in thioredoxins. We showed that the substitution of Asp24 with a hydrophobic residue (D24A) was not sufficient to completely close the cavity. The dynamics of the D24A mutant of yTrx1 at multiple time scales revealed that the D24A mutant presents motions at different time scales near the active site, interaction loops, and water cavity, revealing the existence of a smaller dissected cavity. Molecular dynamics simulation, along with experimental molecular dynamics, allowed a detailed description of the water cavity in wild-type yTrx1 and D24A. The cavity connects the interacting loops, the central ß-sheet, and α-helices 2 and 4. It is formed by three contiguous lobes, which we call lobes A-C. Lobe A is hydrophilic and the most superficial. It is formed primarily by the conserved Lys54. Lobe B is the central lobe formed by the catalytically important residues Cys33 and Asp24, which are strategically positioned. Lobe C is the most hydrophobic and is formed by the conserved cis-Pro73. The central lobe B is closed upon introduction of the D24A mutation, revealing that independent forces other than solvation of Asp24 maintain lobes A and C in the open configuration. These data allow us to better understand the properties of this enzyme.
Assuntos
Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Peroxirredoxinas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Peroxirredoxinas/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
The D216H polymorphism (rs1801968) in TOR1A has been suggested as a risk factor for developing primary dystonia in German subjects not carrying the deletion c.904-906delGAG (∆GAG). However, this association could not be confirmed in other populations with different ethnic backgrounds. The purpose of this study is to evaluate the D216H polymorphism in an Argentinean cohort of 40 patients with primary dystonia and 200 unrelated control subjects. The authors could observe a significantly higher frequency of the H216 variant in dystonic patients lacking ∆GAG as compared with controls.