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1.
Chem Commun (Camb) ; 55(60): 8880-8883, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31321399

RESUMO

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.


Assuntos
Hidrolases/química , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Butiratos/química , Catálise , Cobre/química , Histidina/química , Histidina/genética , Hidrolases/genética , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/química , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Treonina/química , Zinco/química
2.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 337-348, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30595160

RESUMO

The discovery of significant amounts of metabolically active brown adipose tissue (BAT) in adult humans renders it a promising target for anti-obesity therapies by inducing weight loss through increased energy expenditure. The components of the N-acetylaspartate (NAA) pathway are highly abundant in BAT. Aspartate N-acetyltransferase (Asp-NAT, encoded by Nat8l) synthesizes NAA from acetyl-CoA and aspartate and increases energy expenditure in brown adipocytes. However, the exact mechanism how the NAA pathway contributes to accelerated mobilization and oxidation of lipids and the physiological regulation of the NAA pathway remained elusive. Here, we demonstrate that the expression of NAA pathway genes corresponds to nutrient availability and specifically responds to changes in exogenous glucose. NAA is preferentially produced from glucose-derived acetyl-CoA and aspartate and its concentration increases during adipogenesis. Overexpression of Nat8l drains glucose-derived acetyl-CoA into the NAA pool at the expense of cellular lipids and certain amino acids. Mechanistically, we elucidated that a combined activation of neutral and lysosomal (acid) lipolysis is responsible for the increased lipid degradation. Specifically, translocation of the transcription factor EB to the nucleus activates the biosynthesis of autophagosomes and lysosomes. Lipid degradation within lysosomes accompanied by adipose triglyceride lipase-mediated lipolysis delivers fatty acids for the support of elevated mitochondrial respiration. Together, our data suggest a crucial role of the NAA pathway in energy metabolism and metabolic adaptation in BAT.


Assuntos
Adipócitos Marrons/metabolismo , Ácido Aspártico/análogos & derivados , Nutrientes/metabolismo , Acetilcoenzima A/metabolismo , Acetiltransferases/metabolismo , Adipócitos Marrons/fisiologia , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo Marrom/metabolismo , Animais , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Ácido Aspártico/fisiologia , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Oxirredução
3.
Mutat Res ; 814: 7-14, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30659944

RESUMO

For night blindness, a detailed structural exploration of the interactions among G-protein receptor rhodopsin, transducin and arrestin was performed. Rhodopsin is responsible for dim light vision while a point mutation (G90→D90) results in an adverse change in its photo-transduction. The validated 3D models of the three proteins were utilized, and upon mutation and interactions, rhodopsin attained higher stability (evaluated through thermodynamic energy calculations, electrostatic surface potential and solvent accessible area), thereby participating strongly with transducin. Conformational switches in mutated rhodopsin also depicted a firm conformation with few 310 helices accompanied by increased percentage of pure α-helices and sheets. All evaluations were corroborated through paired T-tests. Glu33 (glycosylated unit in the N-terminal zone) of rhodopsin plays a chief role in the overall interaction pattern. Arg69 and Glu33 from wild-type rhodopsin participated in ionic interactions, while the latter set of ionic interaction remained preserved even after mutation. Cys323 (C-terminal residue) and Arg69 formed H-bonds from the wild-type rhodopsin. Cys323 exceptionally supports cellular signaling pattern in the non-mutated situation and for the non-sufferers of night-blindness. Ser297 and Tyr43 from mutated rhodopsin reside in helices and interact with Thr32 of transducin, preserving the steady conformation in activated interacted state, even in the dark. Ser297 lies adjoined to Lys296 (retinal attachment site), which resides in NPXXY motif (an "activation switch" for signal transduction). Thus, the molecular facet for involvement of photo-transduction, which holds a paramount zone in ophthalmology, was dealt with. This might instigate the future prospect for drug discovery to prevent such mutations.


Assuntos
Adaptação à Escuridão/genética , Cegueira Noturna/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas/genética , Rodopsina/genética , Substituição de Aminoácidos , Ácido Aspártico/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Glicina/genética , Humanos , Modelos Moleculares , Cegueira Noturna/metabolismo , Ligação Proteica/genética , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína/genética , Rodopsina/química , Rodopsina/metabolismo , Transdução de Sinais/genética , Relação Estrutura-Atividade
4.
Virulence ; 9(1): 1112-1125, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30067143

RESUMO

Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1ß, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO. ABBREVIATIONS: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-ß-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin-Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1ß: interleukin-1ß; IFN-γ: interferon-γ; TGF-ß: transforming growth factor-ß; ELISA: enzyme-linked immunosorbent assay.


Assuntos
Actinomycetaceae/genética , Arginina/genética , Ácido Aspártico/genética , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Inflamação/induzido quimicamente , Actinomycetaceae/química , Actinomycetaceae/patogenicidade , Substituição de Aminoácidos , Animais , Arginina/química , Ácido Aspártico/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Bovinos , Linhagem Celular , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/ultraestrutura , Feminino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Hemólise , Interleucina-10/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Ovinos , Fator de Necrose Tumoral alfa/genética , Virulência
5.
Medicine (Baltimore) ; 97(28): e11397, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29995784

RESUMO

The frequency of some Cystic Fibrosis (CF) Transmembrane Conductance Regulator gene (CFTR) mutations varies between populations. Genetic testing during newborn screening (NBS) for CF can identify less common mutations with low clinical expression in childhood and previously considered mild but not fully characterized, such as the mutation p.Val232Asp (c.695T > A). The aim of this study was to describe CF patients with the V232D mutation. We identify CF children with the V232D mutation detected by NBS and compare them with CF adults with this mutation whose diagnosis was prompted by clinical symptoms in the same period. We studied clinical, biochemical, spirometric, and prognostic features in both populations. NBS program tested 276,523 children during a period of 14 years (2003-2017) and identified 54 cases of CF. Six children (11%) had the V232D mutation. Over the same period, 5 adults (age 37.6 ±â€Š16.29 years old) with symptoms of CF and this mutation were also diagnosed. Follow-up duration was mean 10.1 years for adults and mean 6.5 years for children. In the adult group, lung function was impaired at diagnosis in all patients (Forced Expiratory Volume1-FEV1-67.12% ±â€Š13.09) and worsened in children tested during evolution (FEV1first: 113%; FEV1last: 64%). Pancreatic insufficiency was present in adult group, with recurrent pancreatitis in 1 present. Although with less clinical expression in children, V232D is associated with pulmonary and pancreatic involvement during adulthood and CF cannot be considered mild. This mutation is present in 11% of all patients diagnosed with CF in our region. Its inclusion in some NBS programs should be taken into account in order to improve the prognosis of affected children.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Adulto , Idoso , Substituição de Aminoácidos , Ácido Aspártico/genética , Fibrose Cística/diagnóstico , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/genética , Feminino , Volume Expiratório Forçado , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação , Triagem Neonatal , Valina/genética
6.
Proc Natl Acad Sci U S A ; 115(32): 8203-8208, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038023

RESUMO

Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1-R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum.


Assuntos
Ácido Aspártico/química , Ativação do Canal Iônico , Domínios Proteicos , Superfamília Shaker de Canais de Potássio/química , Potenciais de Ação , Sequência de Aminoácidos/genética , Animais , Arginina/química , Arginina/genética , Arginina/metabolismo , Ácido Aspártico/genética , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Osmose , Técnicas de Patch-Clamp , Superfamília Shaker de Canais de Potássio/genética , Eletricidade Estática , Água/química , Xenopus
7.
Biochem Biophys Res Commun ; 501(2): 458-464, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29738773

RESUMO

PerR is a metal-dependent peroxide sensing transcription factor which controls the expression of genes involved in peroxide resistance. The function of Bacillus subtilis PerR is mainly dictated by the regulatory metal ion (Fe2+ or Mn2+) coordinated by three N-donor ligands (His37, His91, and His93) and two O-donor ligands (Asp85 and Asp104). While H2O2 sensing by PerR is mediated by Fe2+-dependent oxidation of N-donor ligand (either His37 or His91), one of the O-donor ligands (Asp104), but not Asp85, has been proposed as the key residue that regulates the sensitivity of PerR to H2O2. Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and H2O2 sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of H2O2 in the presence of excess Fe2+ sufficient for the formation of the Fe2+-bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe2+, suggesting that Fe2+ is not an effective corepressor for this mutant protein in vivo. Furthermore, in vitro metal titration assays indicate that D104E-PerR has a significantly reduced affinity for Fe2+, but not for Mn2+, when compared to wild type PerR. These data indicate that the type of O-donor ligand (Asp vs. Glu) at position 104 is an important determinant in providing high Fe2+-binding affinity required for the sensing of the physiologically relevant Fe2+-levels, in addition to its role in rendering PerR highly sensitive to physiological levels of H2O2. In comparison, the D85E-PerR did not show a perturbed change in Fe2+-binding affinity, however, it displayed a slightly decreased sensitivity to H2O2 both in vivo and in vitro, suggesting that the type of O-donor ligand (Asp vs. Glu) at position 85 may be important for the fine-tuning of H2O2 sensitivity.


Assuntos
Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Proteínas Repressoras/metabolismo , Substituição de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Polarização de Fluorescência , Ligantes , Oxirredução , Oxigênio/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
Proc Natl Acad Sci U S A ; 115(13): E2921-E2929, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531047

RESUMO

Most replicative DNA polymerases (DNAPs) are endowed with a 3'-5' exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp121 of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp90 in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.


Assuntos
Ácido Aspártico/genética , Fagos Bacilares/enzimologia , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação , Sequência de Aminoácidos , Fagos Bacilares/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Mutagênese Sítio-Dirigida , Homologia de Sequência
9.
Biochem Biophys Res Commun ; 496(1): 101-104, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29305262

RESUMO

The muscarinic M2 receptor (M2R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M2R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M2R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M2R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M2R.


Assuntos
Acetilcolina/administração & dosagem , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Animais , Ácido Aspártico/genética , Células Cultivadas , Sequência Conservada , Relação Dose-Resposta a Droga , Mutagênese Sítio-Dirigida , Oócitos , Mutação Puntual/genética , Receptor Muscarínico M2/efeitos dos fármacos , Relação Estrutura-Atividade , Xenopus laevis
10.
J Alzheimers Dis ; 61(1): 41-46, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29103041

RESUMO

A new risk gene associated with amyotrophic lateral sclerosis (ALS) has recently been identified: the Tank-binding kinase 1 (TBK1) gene. Up to now, 90 TBK1 variants have been described in ALS patients with or without frontotemporal dementia (FTD), thus making TBK1 the third or fourth most frequent genetic cause of ALS and FTD. A point mutation analysis in a cohort of 69 Italian ALS patients was performed in order to analyze the frequency of TBK1 mutations and the correlation with clinical phenotypes. The analysis identified the novel variant p.Tyr424Asp in a patient with a rapid progression of the disease. Our data supports the implication of TBK1 in ALS pathogenesis in Italy.


Assuntos
Esclerose Amiotrófica Lateral/genética , Disfunção Cognitiva/genética , Predisposição Genética para Doença/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Idoso , Esclerose Amiotrófica Lateral/complicações , Esclerose Amiotrófica Lateral/diagnóstico por imagem , Ácido Aspártico/genética , Proteína C9orf72/genética , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/etiologia , Análise Mutacional de DNA , Feminino , Fluordesoxiglucose F18/metabolismo , Estudos de Associação Genética , Humanos , Itália , Masculino , Testes de Estado Mental e Demência , Pessoa de Meia-Idade , Testes Neuropsicológicos , Tomografia por Emissão de Pósitrons , Tirosina/genética
11.
Gene ; 641: 355-360, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29080836

RESUMO

BACKGROUND: Hypochondroplasia (HCH; OMIM 146000) is a common autosomal dominant skeletal dysplasia characterized by disproportionate short stature, short extremities, relative macrocephaly, and lumbar lordosis. Because of its clinical and genetic heterogeneity, gene mutational analysis is particularly important in diagnosis and the phenotypes may be ameliorated if diagnosed early. MATERIALS AND METHODS: In this study, we examined a Chinese family with HCH, performed an inductive analysis of their clinical features and radiographic results, and applied targeted exome sequencing (TES) technology to perform a molecular diagnosis. RESULTS: The proband and his mother all presented disproportionate short stature, short, stubby extremities, unchanged interpedicular distances from L1-L5, and short iliac bones, with a 'fish mouth-shaped' sciatic notch. The mother received induced abortion recently because an ultrasound showed short femur length of her fetus at 24-week gestation. Eventually, a novel heterozygous mutation (c.1145G>A) in FGFR3 was identified by TES in the proband, his mother, and her fetus; this causes the substitution of glycine with aspartic acid in codon 382. CONCLUSIONS: In this study, we diagnosed a Chinese pedigree with HCH based on clinical data, radiographic features, and genetic testing results. Our results extend the genetic mutation spectrum of FGFR3 and demonstrate that TES is an effective method for the diagnosis of skeletal dysplasia in clinical practices.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Osso e Ossos/anormalidades , Nanismo/diagnóstico , Nanismo/genética , Deformidades Congênitas dos Membros/diagnóstico , Deformidades Congênitas dos Membros/genética , Lordose/diagnóstico , Lordose/genética , Mutação/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Adulto , Ácido Aspártico/genética , Criança , Exoma/genética , Glicina/genética , Heterozigoto , Humanos , Masculino , Patologia Molecular/métodos , Linhagem
12.
Gene ; 641: 226-234, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29066301

RESUMO

Mycobacterium tuberculosis katG gene is responsible for production of an enzyme catalase peroxidase that peroxidises and activates the prodrug Isoniazid (INH), a first-line antitubercular agent. INH interacts with catalase peroxidase enzyme within its heme pocket and gets converted to an active form. Mutations occurring in katG gene are often linked to reduced conversion rates for INH. This study is focussed on one such mutation occurring at residue 279, where glycine often mutates to aspartic acid (G279D). In the present study, several structural analyses were performed to study the effect of this mutation on functionality of KatG protein. On comparison, mutant protein exhibited a lower docking score, smaller binding cavity and reduced affinity towards INH. Molecular dynamics analysis revealed the mutant to be more rigid and less compact than the native protein. Essential dynamics analysis determined correlated motions of residues within the protein structure. G279D mutant was found to have many residues that showed related motions and an undesirable effect on the functionality of protein.


Assuntos
Catalase/genética , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Isoniazida/farmacologia , Mutação/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Peroxidase/genética , Ácido Aspártico/genética , Glicina/genética , Simulação de Dinâmica Molecular , Proteínas Mutantes/genética
13.
Toxins (Basel) ; 9(12)2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29189743

RESUMO

Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α-helical content to CaMWt, and only modest differences in α-helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C-terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N-terminal CaM and CyaA-ACD.


Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Toxina Adenilato Ciclase/genética , Alanina/genética , Alanina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Calmodulina/genética , Dicroísmo Circular , Mutagênese Sítio-Dirigida , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
14.
Mol Biol (Mosk) ; 51(5): 831-835, 2017 Sep-Oct.
Artigo em Russo | MEDLINE | ID: mdl-29116070

RESUMO

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.


Assuntos
Escherichia coli K12/química , Proteínas de Escherichia coli/química , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Mutação de Sentido Incorreto , Domínios Proteicos
15.
BMC Med Genet ; 18(1): 116, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29047356

RESUMO

BACKGROUND: Mutations in LMNA gene, encoding Lamin A/C, cause a diverse array of phenotypes, collectively referred to as laminopathies. The most common manifestation is dilated cardiomyopathy (DCM), occurring in conjunction with variable skeletal muscle involvement but without involvement of the coronary arteries. Much less commonly, LMNA mutations cause progeroid syndromes, whereby an early-onset coronary artery disease (CAD) is the hallmark of the disease. We report a hitherto unreported compound cardiac phenotype, dubbed as "non-syndromic cardiac progeria", in a young patient who carried a rare pathogenic variant in the LMNA gene and developed progressive degeneration of various cardiac structures, as seen in the elderly. The phenotype resembled the progeroid syndromes, except that it was restricted to the heart and did not involve other organs. CASE PRESENTATION: The patient was a well-developed Caucasian female who presented at age 29 years with an acute myocardial infarction (MI) and was found to have extensive CAD. She had none of the conventional risk factors for atherosclerosis. She underwent coronary artery bypass surgery but continued to require multiple percutaneous coronary interventions for symptomatic obstructive coronary lesions. During the course of next 10 years, she developed mitral regurgitation, degenerative mitral and aortic valve diseases, atrial flutter, and progressive conduction defects. She died from progressive heart failure with predominant involvement of the right ventricle and severe tricuspid regurgitation. Cardiac phenotype in this young patient resembled degenerative cardiac diseases of the elderly and the progeroid syndromes. However, in contrast to the progeroid syndromes, the phenotype was restricted to the heart and did not involve other organs. Thus, the phenotype was dubbed as a non-syndromic cardiac progeria. Genetic screening of several cardiomyopathy genes, including LMNA, which is a causal gene for progeroid syndromes, led to identification of a very rare pathogenic p.Asp300Asn variant in the LMNA gene. CONCLUSIONS: We infer that the LMNA p.Asp300Asn mutation is pathogenic in non-syndromic cardiac progeria. Mutations involving codon 300 in the LMNA gene have been associated with progeroid syndromes involving multiple organs. Collectively, the data provide credence to the causal role of p.Asp300Asn mutation in the pathogenesis of non-syndromic cardiac progeria.


Assuntos
Lamina Tipo A/genética , Mutação de Sentido Incorreto , Progéria/genética , Adulto , Sequência de Aminoácidos , Asparagina/genética , Ácido Aspártico/genética , Sequência de Bases , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/fisiopatologia , Ecocardiografia/métodos , Eletrocardiografia/métodos , Evolução Fatal , Feminino , Humanos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Linhagem , Progéria/diagnóstico , Progéria/fisiopatologia , Homologia de Sequência de Aminoácidos
16.
New Phytol ; 216(3): 814-828, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28722263

RESUMO

Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein 'Sensitive to ABA and Drought 2' (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11D126N in F. tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11D122N in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.


Assuntos
Fagopyrum/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Arabidopsis/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Citoplasma/metabolismo , Fagopyrum/genética , Teste de Complementação Genética , Mutação , Fenilpropionatos/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Rutina/biossíntese , Rutina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
J Biol Chem ; 292(36): 14775-14785, 2017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28729424

RESUMO

Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.


Assuntos
Proteínas de Transporte de Nucleosídeos/química , Proteínas de Transporte de Nucleosídeos/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mutação , Proteínas de Transporte de Nucleosídeos/genética
18.
Headache ; 57(7): 1136-1144, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28573794

RESUMO

OBJECTIVE: To search for differences in prevalence of a CACNA1E variant between migraine without aura, various phenotypes of migraine with aura, and healthy controls. BACKGROUND: Familial hemiplegic migraine type 1 (FHM1) is associated with mutations in the CACNA1A gene coding for the alpha 1A (Cav 2.1) pore-forming subunit of P/Q voltage-dependent Ca2+ channels. These mutations are not found in the common forms of migraine with or without aura. The alpha 1E subunit (Cav 2.3) is the counterpart of Cav 2.1 in R-type Ca2+ channels, has different functional properties, and is encoded by the CACNA1E gene. METHODS: First, we performed a total exon sequencing of the CACNA1E gene in three probands selected because they had no abnormalities in the three FHM genes. In a patient suffering from basilar-type migraine, we identified a single nucleotide polymorphism (SNP) in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760; Minor Allele Frequency 0.2241) hitherto not studied in migraine. In a second step, we determined its occurrence in four groups by direct sequencing on blood genomic DNA: migraine patients without aura (N = 24), with typical aura (N = 55), complex neurological auras (N = 19; hemiplegic aura: N = 15; brain stem aura: N = 4), and healthy controls (N = 102). RESULTS: The Asp859Glu - rs35737760 SNP of the CACNA1E gene was present in 12.7% of control subjects and in 20.4% of the total migraine group. In the migraine group it was significantly over-represented in patients with complex neurological auras (42.1%), OR 4.98 (95% CI: 1.69-14.67, uncorrected P = .005, Bonferroni P = .030, 2-tailed Fisher's exact test). There was no significant difference between migraine with typical aura (10.9%) and controls. CONCLUSIONS: We identified a polymorphism in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760) that is more prevalent in hemiplegic and brain stem aura migraine. This missense variant causes a change from aspartate to glutamate at position 859 of the Cav 2.3 protein and might modulate the function of R-type Ca2+ channels. It could thus be relevant for migraine with complex neurological aura, although this remains to be proven.


Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Ataxia Cerebelar/genética , Predisposição Genética para Doença/genética , Transtornos de Enxaqueca/genética , Polimorfismo de Nucleotídeo Único/genética , Ácido Aspártico/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Éxons/genética , Feminino , Frequência do Gene , Ácido Glutâmico/genética , Humanos , Masculino , Transtornos de Enxaqueca/classificação , Fenótipo , Estudos Retrospectivos , Estatísticas não Paramétricas
19.
ACS Synth Biol ; 6(9): 1635-1641, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28548828

RESUMO

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X)2DP, AE(X)3DP, AE(X)4DP, AE(X)5DP, and AE(X)6DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X)2DP, AE(X)3DP, and AE(X)4DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.


Assuntos
Ácido Aspártico/química , Bacteriófago M13/genética , Ouro/química , Nanopartículas Metálicas/química , Biblioteca de Peptídeos , Prolina/química , Mapeamento de Interação de Proteínas/métodos , Ácido Aspártico/genética , Sítios de Ligação , Prolina/genética , Ligação Proteica , Análise de Sequência de Proteína/métodos , Coloração e Rotulagem/métodos
20.
Biochemistry (Mosc) ; 82(1): 46-59, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28320286

RESUMO

The plasma membrane Pma1 H+-ATPase of the yeast Saccharomyces cerevisiae contains conserved residue Asp739 located at the interface of transmembrane segment M6 and the cytosol. Its replacement by Asn or Val (Petrov et al. (2000) J. Biol. Chem., 275, 15709-15716) or by Ala (Miranda et al. (2011) Biochim. Biophys. Acta, 1808, 1781-1789) caused complete blockage of biogenesis of the enzyme, which did not reach secretory vesicles. It was proposed that a strong ionic bond (salt bridge) could be formed between this residue and positively charged residue(s) in close proximity, and the replacement D739A disrupted this bond. Based on a 3D homology model of the enzyme, it was suggested that the conserved Arg811 located in close proximity to Asp739 could be such stabilizing residue. To test this suggestion, single mutants with substituted Asp739 (D739V, D739N, D739A, and D739R) and Arg811 (R811L, R811M, R811A, and R811D) as well as double mutants carrying charge-neutralizing (D739A/R811A) or charge-swapping (D739R/R811D) substitutions were used. Expression of ATPases with single substitutions R811A and R811D were 38-63%, and their activities were 29-30% of the wild type level; ATP hydrolysis and H+ transport in these enzymes were essentially uncoupled. For the other substitutions including the double mutations, the biogenesis of the enzyme was practically blocked. These data confirm the important role of Asp739 and Arg811 residues for the biogenesis and function of the enzyme, suggesting their importance for defining H+ transport determinants but ruling out, however, the existence of a strong ionic bond (salt bridge) between these two residues and/or importance of such bridge for structure-function relationships in Pma1 H+-ATPase.


Assuntos
Modelos Moleculares , ATPases Translocadoras de Prótons/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , Arginina/química , Arginina/genética , Ácido Aspártico/química , Ácido Aspártico/genética , Mutação de Sentido Incorreto , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
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