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1.
Nat Commun ; 11(1): 1755, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273511

RESUMO

Asparagine synthetase (ASNS) catalyses the ATP-dependent conversion of aspartate to asparagine. However, both the regulation and biological functions of asparagine in tumour cells remain largely unknown. Here, we report that p53 suppresses asparagine synthesis through the transcriptional downregulation of ASNS expression and disrupts asparagine-aspartate homeostasis, leading to lymphoma and colon tumour growth inhibition in vivo and in vitro. Moreover, the removal of asparagine from culture medium or the inhibition of ASNS impairs cell proliferation and induces p53/p21-dependent senescence and cell cycle arrest. Mechanistically, asparagine and aspartate regulate AMPK-mediated p53 activation by physically binding to LKB1 and oppositely modulating LKB1 activity. Thus, we found that p53 regulates asparagine metabolism and dictates cell survival by generating an auto-amplification loop via asparagine-aspartate-mediated LKB1-AMPK signalling. Our findings highlight a role for LKB1 in sensing asparagine and aspartate and connect asparagine metabolism to the cellular signalling transduction network that modulates cell survival.


Assuntos
Asparagina/metabolismo , Ácido Aspártico/metabolismo , Neoplasias do Colo/metabolismo , Homeostase , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Asparagina/genética , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Ácido Aspártico/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/terapia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
2.
J Clin Neurosci ; 75: 223-225, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32223976

RESUMO

About 10% of Amyotrophic Lateral Sclerosis (ALS) cases are familial (FALS), mainly related to mutations in C9ORF72, SOD1, TARDBP, and FUS genes. Recent data revealed the presence of multiple variants in ALS-associated genes in FALS in excess of what is to be expected by chance. FALS patients not carrying a pathogenic genetic mutation detected in their kindred have been reported. We report a FALS case, who did not carry the p.Ala5Val heterozygous SOD1 mutation that had been detected in other affected subjects of his kindred. He underwent Next-Generation Sequencing, revealing a novel p.Glu46Asp heterozygous OPTN variant of uncertain significance (VUS). Discordant genetic test results in FALS cases within the same family and the detection of variants of uncertain significance increase the complexities of genetic counselling.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico , Esclerose Amiotrófica Lateral/genética , Proteínas de Ciclo Celular/genética , Variação Genética/genética , Proteínas de Membrana Transportadoras/genética , Mutação de Sentido Incorreto/genética , Superóxido Dismutase-1/genética , Alanina/genética , Ácido Aspártico/genética , Feminino , Testes Genéticos/métodos , Glutamina/genética , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Valina/genética
3.
BMC Med Genet ; 21(1): 60, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209048

RESUMO

BACKGROUND: Brachydactyly type A1(BDA-1) is an autosomal dominant disorder which is caused by heterozygous pathogenic variants in a specific region of the N-terminal active fragment of Indian Hedgehog (IHH). The disorder is mainly characterized by shortening or missing of the middle phalanges. In this study, Our purpose is to identify the pathogenic variations associated with BDA-1 involved in a five-generation Chinese family. METHODS: A BDA-1 family with 8 affected and 14 unaffected family members was recruited. Whole exome sequencing (WES) was performed to identify the pathogenic variant in the proband, and which was later confirmed and segregated by Sanger sequencing. The significance of variants were assessed using several molecular and bioinformatics analysis methods. RESULTS: We uncovered a novel heterozygous missense variant c.299A > G (p.D100G) at the mutational hotspot of IHH gene following whole-exome sequencing of a Chinese family with BDA-1. The variant co-segregated with BDA-1 in the pedigree, showed 100% penetrance for phalange phenotype with variable expressivity. CONCLUSIONS: In conclusion, this study reports a five-generation Chinese family with BDA-1 due to a novel pathogenic variant (c.299A > G (p.D100G)) of IHH and expands the clinical and genetic spectrum of BDA-1.


Assuntos
Braquidactilia/genética , Proteínas Hedgehog/genética , Mutação de Sentido Incorreto , Adulto , Substituição de Aminoácidos , Ácido Aspártico/genética , Braquidactilia/diagnóstico , Braquidactilia/patologia , China , Análise Mutacional de DNA , Família , Feminino , Predisposição Genética para Doença , Glicina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma , Adulto Jovem
4.
Microbiol Res ; 230: 126352, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634726

RESUMO

Rhoptry protein 18 (ROP18) is a major determinant of strain-specific virulence in Toxoplasma gondii. The kinase activity of ROP18 is required for acute virulence, while the aspartate in the catalytic loop of ROP18 is considered essential for phosphoryl transfer. We showed that a single amino acid mutation at the catalytic aspartate residue (D409A mutation) significantly altered ROP18 kinase activity in vitro, and abolished ROP18-mediated ATF6ß degradation. Furthermore, the investigated single amino acid mutation in ROP18 led to alternation of subcellular localization of ROP18 protein. Our findings demonstrate that a single amino acid mutation on the proton transport catalytic aspartic acid induced alternations associated with ROP18 protein.


Assuntos
Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/enzimologia , Motivos de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Mutação de Sentido Incorreto , Transporte Proteico , Proteínas Serina-Treonina Quinases/química , Prótons , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/metabolismo
5.
Biochim Biophys Acta Gen Subj ; 1864(1): 129450, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676296

RESUMO

BACKGROUND: Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs. METHODS: Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3. RESULTS: This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 µM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 µM vs 63 µM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10-5 µm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10-3 µm2/s). CONCLUSIONS: We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs. GENERAL SIGNIFICANCE: Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.


Assuntos
Adesão Celular/genética , Quinase 1 de Adesão Focal/genética , Adesões Focais/genética , Conformação Proteica , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Ácido Aspártico/genética , Sítios de Ligação/genética , Movimento Celular/genética , Quinase 1 de Adesão Focal/química , Adesões Focais/química , Regulação da Expressão Gênica/genética , Humanos , Lisina/química , Lisina/genética , Células MCF-7 , Proteínas de Membrana/química , Proteínas de Membrana/genética , Paxilina/química , Paxilina/genética , Ligação Proteica/genética , Engenharia de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Vinculina/química , Vinculina/genética
6.
J Mol Model ; 26(1): 1, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31834477

RESUMO

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/genética
7.
Int J Mol Sci ; 20(24)2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31835305

RESUMO

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT "supermutant" of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.


Assuntos
Flavina-Adenina Dinucleotídeo/química , Mutação de Sentido Incorreto , Nucleotidiltransferases/química , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
8.
J Neuromuscul Dis ; 6(4): 485-501, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31594244

RESUMO

BACKGROUND: Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed. OBJECTIVE: A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause. METHODS: Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed. RESULTS: Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress. CONCLUSIONS: This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/genética , Ácido Aspártico/genética , Ácido Glutâmico/genética , Mitocôndrias/genética , Mutação/genética , Animais , Ácido Aspártico/metabolismo , Dermatomiosite/metabolismo , Cães , Ácido Glutâmico/metabolismo , Humanos , Mitocôndrias/metabolismo , Miosite/genética , Oxirredução , Polimiosite/metabolismo
9.
J Immunol ; 203(7): 1918-1929, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31484733

RESUMO

ESAT-6 is a small secreted protein of Mycobacterium tuberculosis involved in the ESAT-6 secretion system (ESX-1)-mediated virulence and pathogenesis. The protein interacts with ß2M, causing downregulation of MHC class I Ag presentation, which could be one of the mechanisms by which it favors increased survival of the bacilli inside the host. In an earlier study, we have shown that the C-terminal region of ESAT-6 is crucial for its interaction with ß2M. However, the interface of ß2M involved in interaction with ESAT-6 and detailed physicochemical changes associated with ESAT-6:ß2M complexation are not fully defined. In this study, using computational and site-directed mutagenesis studies, we demonstrate the presence of strong noncovalent hydrophobic interactions between ESAT-6 and ß2M in addition to the vital hydrogen bonding between the aspartate residue (Asp53) of ß2M and methionine (Met93) of ESAT-6. Docking-based high-throughput virtual screening followed by 16-point screening on microscale thermophoresis resulted in the identification of two potent inhibitors (SM09 and SM15) that mask the critical Met93 residue of ESAT-6 that is required for ESAT-6:ß2M interaction and could rescue cell surface expression of ß2M and HLA in human macrophages as well as MHC class I Ag presentation suppressed by ESAT-6 in peritoneal macrophages isolated from C57BL/6 mice. Both SM09 and SM15 significantly inhibited intracellular survival of M. tuberculosis in human macrophages. Further, we characterized the physicochemical properties involved in the ESAT-6:ß2M complexation, which may help in understanding host-pathogen interactions.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/química , Microglobulina beta-2/química , Substituição de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos Peritoneais/química , Macrófagos Peritoneais/imunologia , Camundongos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/fisiologia , Estrutura Quaternária de Proteína , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia
10.
J Biol Chem ; 294(40): 14574-14590, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31375564

RESUMO

Human tankyrase-1 (TNKS) is a member of the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: the PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity.


Assuntos
Hidroliases/química , Complexos Multiproteicos/química , Estrutura Quaternária de Proteína/genética , Tanquirases/química , Proteínas de Ligação a Telômeros/química , Adenosina Difosfato Ribose/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Repetição de Anquirina/genética , Ácido Aspártico/genética , Sítios de Ligação/genética , Ácido Glutâmico/genética , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tanquirases/genética , Tanquirases/metabolismo , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Via de Sinalização Wnt/genética
11.
Nat Commun ; 10(1): 3357, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350392

RESUMO

Amyloid-ß (Aß) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aß 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt ß-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aß structures, and both self-associate through two distinct interfaces. One of these is a unique Aß interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aß 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aß segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Humanos , Ácido Isoaspártico/genética , Ácido Isoaspártico/metabolismo , Isomerismo , Mutação , Conformação Proteica
12.
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
13.
Metab Eng ; 54: 244-254, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31063790

RESUMO

L-aspartate is an important 4-carbon platform compound that can be used as the precursor of numerous chemical products. The bioproduction of L-aspartate directly from biomass resources is expected to provide a more cost-competitive technique route. Yet little metabolic engineering work on this matter has been carried out. In this study, we designed a shortcut pathway of L-aspartate biosynthesis in Escherichia coli, with a maximized stoichiometric yield of 2 mol/mol glucose. L-aspartate aminotransferase (AspC) was overexpressed for producing L-aspartate and coexpressed with L-aspartate-a-decarboxylase (PanD) for producing L-aspartate's derivative ß-alanine. L-aspartate could only be detected after directing carbon flux towards oxaloacetate and blocking the "futile cycle" with TCA cycle. A cofactor self-sufficient system successfully improved the efficiency of AspC-catalyzing L-aspartate biosynthesis reaction, and the glucose uptake remolding capably decreased byproducts from pyruvate. More targets were modified for relieving the bottleneck during fed-batch bioconversion. As a result, 1.01 mol L-aspartate/mol glucose and 1.52 mol ß-alanine/mol glucose were produced in corresponding strains respectively. Fed-batch bioconversion allowed 249 mM (33.1 g/L) L-aspartate or 424 mM (37.7 g/L) ß-alanine production, respectively. The study provides a novel promising metabolic engineering route for the production of L-aspartate and its derivate chemicals using biomass resources. These results also represent the first report of the efficient bioproduction of L-aspartate directly from glucose in E. coli and the highest yield of ß-alanine reported so far.


Assuntos
Ácido Aspártico , Carboxiliases , Ciclo do Ácido Cítrico/genética , Proteínas de Escherichia coli , Escherichia coli , Engenharia Metabólica , beta-Alanina , Ácido Aspártico/biossíntese , Ácido Aspártico/genética , Carboxiliases/genética , Carboxiliases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Alanina/genética , beta-Alanina/metabolismo
14.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 722-731, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100408

RESUMO

Kynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5'-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and ß-benzoyl-L-alanine, but not O-benzoyl-l-serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with ß-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with ß-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The 1H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold reduction of kcat and 30-fold reduction in kcat/Km with L-kynurenine, but very little effect on the reaction of O-benzoyl-l-serine. Thus, the rate-determining step in the reaction of S36A Pfkynase is the Cß-Cγ bond cleavage. These results support the hypothesis that Ser-36 together with Tyr-226 is part of an oxyanion hole that polarizes the carbonyl of the substrate in the catalytic mechanism of Pfkynase.


Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Pseudomonas fluorescens/enzimologia , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Proteínas de Bactérias/genética , Hidrolases/genética , Mutação de Sentido Incorreto , Pseudomonas fluorescens/genética , Serina/química , Serina/genética
15.
Ann Biol Clin (Paris) ; 77(3): 287-294, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31021322

RESUMO

Biology flourished during the XXth century and was profoundly disrupted during the last decade because of the transition to the post-genomic era, the spread of high-throughput biology, and the advent of a relatively new discipline, namely bioinformatics. This latter, which encompasses the collection, organization and analysis of biological data using the computer tool, has quickly become inseparable from the studies related to the genome understanding. The consequences of the different mutations that may affect our genes are responsible for a change in the protein sequence and are likely to affect, for example, the stability of the protein, its intracellular targeting, its maturation, its assembly in a multimeric structure, the essential sites for its enzymatic activity or for the interaction with ligands. Thus, a number of bioinformatic developments have made it possible to set up in silico prediction tools of the structure of a protein that is aiming at predicting the impact of local mutations on the structure of proteins. Throughout our study, we have been interested in exploring, through in silico bioinformatic study, three analytical, prediction and modeling, software, choosing as exemple the G12D mutation that affects the proto-oncogene KRAS found in numerous algerian patients with bronchopulmonary cancers cells (NSCLC). This study allowed us to integrate these bioinformatic tools into our laboratory of developmental biology and LBDD differentiation at the University of Oran 1 Ahmed Benbella, in Algeria. Thus, we have been able to conclude, even if the found mutation is predicted to be tolerated and has no deleterious effect on the entire Ras protein, that the consequence of this missense mutation depends mainly on the position in the protein and the chemical properties of the amino acid involved in the substitution and which shows a strong affinity with the GTP molecule.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Biologia Computacional/métodos , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras)/genética , Argélia , Substituição de Aminoácidos/genética , Ácido Aspártico/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Éxons , Estudos de Associação Genética/métodos , Glicina/genética , Humanos , Neoplasias Pulmonares/patologia , Modelos Moleculares , Proteínas Proto-Oncogênicas p21(ras)/química , Software
16.
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
17.
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
18.
FEBS J ; 286(6): 1204-1213, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30657257

RESUMO

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site-directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure-induced reactivation was due to the decrease in the volume of the system, -ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme.


Assuntos
Ácido Aspártico/metabolismo , Bactérias/enzimologia , Di-Hidro-Orotase/química , Di-Hidro-Orotase/metabolismo , Zinco/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Domínio Catalítico , Cristalografia , Di-Hidro-Orotase/genética , Mutagênese Sítio-Dirigida , Mutação , Pressão , Conformação Proteica
19.
Proteins ; 87(1): 34-40, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30315603

RESUMO

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing KD values from 625 ± 118 to 468 ± 13 µM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.


Assuntos
Ácido Aspártico/metabolismo , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Sefarose/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Especificidade por Substrato
20.
Proc Natl Acad Sci U S A ; 115(42): E9782-E9791, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30279180

RESUMO

Human genetic studies have given evidence of familial, disease-causing mutations in the analogous amino acid residue shared by three related RNA binding proteins causative of three neurological diseases. Alteration of aspartic acid residue 290 of hnRNPA2 to valine is believed to predispose patients to multisystem proteinopathy. Mutation of aspartic acid 262 of hnRNPA1 to either valine or asparagine has been linked to either amyotrophic lateral sclerosis or multisystem proteinopathy. Mutation of aspartic acid 378 of hnRNPDL to either asparagine or histidine has been associated with limb girdle muscular dystrophy. All three of these aspartic acid residues map to evolutionarily conserved regions of low-complexity (LC) sequence that may function in states of either intrinsic disorder or labile self-association. Here, we present a combination of solid-state NMR spectroscopy with segmental isotope labeling and electron microscopy on the LC domain of the hnRNPA2 protein. We show that, for both the wild-type protein and the aspartic acid 290-to-valine mutant, labile polymers are formed in which the LC domain associates into an in-register cross-ß conformation. Aspartic acid 290 is shown to be charged at physiological pH and immobilized within the polymer core. Polymers of the aspartic acid 290-to-valine mutant are thermodynamically more stable than wild-type polymers. These observations give evidence that removal of destabilizing electrostatic interactions may be responsible for the increased propensity of the mutated LC domains to self-associate in disease-causing conformations.


Assuntos
Ácido Aspártico/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Mutação , Polímeros/química , Sequência de Aminoácidos , Ácido Aspártico/genética , Humanos , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos
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