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1.
Nat Commun ; 10(1): 2574, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189925

RESUMO

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Domínios Proteicos/genética , Adenosina Trifosfatases/genética , Ácido Glutâmico/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Relação Estrutura-Atividade , Tirosina/genética , Tirosina/metabolismo
2.
Diabetes Res Clin Pract ; 152: 135-145, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31102685

RESUMO

INTRODUCTION: Many studies have evaluated the association of paraoxonase-1 (PON1) gene polymorphisms with enzyme activity and concentration in type 2 diabetes mellitus (T2DM). However, the exact impact of these polymorphisms is not still obvious. Hence, we conducted a systematic review and meta-analysis to clarify the association of PON1 polymorphisms with its enzyme characteristics in T2DM patients and non-diabetic individuals. METHODS: We searched electronic databases including PubMed, Web of Science, Embase and Scopus for publications by April 2018. The pooled response ratio (rr) for the association and their corresponding 95% confidence intervals (CIs) were calculated using a fixed-effect model. RESULTS: Fifteen relevant studies fulfilled our inclusion criteria. The results showed a 1.25-fold increase in total PON1 activity in non-diabetic group against T2DM patients (p-value = 0.024). Also, only Q192R and L55M polymorphisms had sufficient studies to be included in the meta-analysis. All three genotypes of Q192R polymorphism showed significantly different activities between the study groups with the highest pooled effect size for RR genotype (rrQQ < rrQR < rrRR) while this difference was seen only in LL genotype of L55M polymorphism. Therefore, Q192R polymorphism was more correlated with type 2 diabetes mellitus. In case of concentration, there was no significant differences between two groups (p-value = 0.897). CONCLUSION: Current meta-analysis suggested that the observed difference of total PON1 activity was due to the different activity of various genotypes of PON1 enzyme in case of L55M and Q192R polymorphisms so that LL and RR genotypes had the most important role in the establishment of mentioned difference.


Assuntos
Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Diabetes Mellitus Tipo 2/genética , Mutação com Ganho de Função , Polimorfismo Genético , Substituição de Aminoácidos , Arginina/genética , Diabetes Mellitus Tipo 2/metabolismo , Ativação Enzimática/genética , Feminino , Genótipo , Ácido Glutâmico/genética , Humanos , Leucina/genética , Masculino , Metionina/genética
3.
Nat Commun ; 10(1): 1832, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015432

RESUMO

Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity.


Assuntos
Apoptose , Ceramidas/metabolismo , Mitocôndrias/fisiologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Sítios de Ligação/genética , Ceramidas/química , Técnicas de Inativação de Genes , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Simulação de Dinâmica Molecular , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/isolamento & purificação , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/química , Canal de Ânion 2 Dependente de Voltagem/genética , Canal de Ânion 2 Dependente de Voltagem/isolamento & purificação
4.
Appl Microbiol Biotechnol ; 103(10): 4033-4043, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937497

RESUMO

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-L-arginyl-poly-[L-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.


Assuntos
Proteínas de Bactérias/biossíntese , Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/metabolismo , Lisina/metabolismo , Engenharia Metabólica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Glutâmico/genética , Lisina/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Nucleic Acids Res ; 47(3): 1404-1415, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30541105

RESUMO

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine 'toggles' between interacting with a glutamate residue in the 'lid' subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an 'open' state to a 'closed' state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.


Assuntos
Empacotamento do DNA/genética , DNA Viral/genética , Genoma Viral/genética , Montagem de Vírus/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Arginina/genética , Arginina/metabolismo , Bacteriófago lambda/enzimologia , Catálise , Endodesoxirribonucleases/genética , Ácido Glutâmico/genética , Hidrólise , Fosfatos/metabolismo
6.
Biochim Biophys Acta Bioenerg ; 1859(8): 577-590, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29719208

RESUMO

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b595, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b595 and heme b558. It is concluded that 1) O2 binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O2 binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b595, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b595) → E445 (heme d) → oxygenated heme d.


Assuntos
Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Elétrons , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Oxirredutases/metabolismo , Oxigênio/química , Prótons , Respiração Celular , Citocromos/química , Citocromos/genética , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Heme/química , Heme/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Oxirredutases/química , Oxirredutases/genética
7.
Emerg Microbes Infect ; 7(1): 68, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29691362

RESUMO

Understanding Zika virus infection dynamics is essential, as its recent emergence revealed possible devastating neuropathologies in humans, thus causing a major threat to public health worldwide. Recent research allowed breakthrough in our understanding of the virus and host pathogenesis; however, little is known on its impact on its main vector, Aedes aegypti. Here we show how Zika virus targets Aedes aegypti's neurons and induces changes in its behavior. Results are compared to dengue virus, another flavivirus, which triggers a different pattern of behavioral changes. We used microelectrode array technology to record electrical spiking activity of mosquito primary neurons post infections and discovered that only Zika virus causes an increase in spiking activity of the neuronal network. Confocal microscopy also revealed an increase in synapse connections for Zika virus-infected neuronal networks. Interestingly, the results also showed that mosquito responds to infection by overexpressing glutamate regulatory genes while maintaining virus levels. This neuro-excitation, possibly via glutamate, could contribute to the observed behavioral changes in Zika virus-infected Aedes aegypti females. This study reveals the importance of virus-vector interaction in arbovirus neurotropism, in humans and vector. However, it appears that the consequences differ in the two hosts, with neuropathology in human host, while behavioral changes in the mosquito vector that may be advantageous to the virus.


Assuntos
Aedes/fisiologia , Comportamento Animal , Neurônios/virologia , Tropismo Viral , Aedes/virologia , Animais , Vírus da Dengue/fisiologia , Fenômenos Eletrofisiológicos , Feminino , Ácido Glutâmico/genética , Humanos , Microeletrodos , Microscopia Confocal , Mosquitos Vetores/virologia , Rede Nervosa/virologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Sinapses/ultraestrutura , Sinapses/virologia , Zika virus/fisiologia , Infecção por Zika virus/virologia
8.
Protein J ; 37(2): 122-131, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29549627

RESUMO

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic ß-lactam resistance. Though PBP5 does not have ß-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A ß-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in ß-lactamases. It is speculated that ß-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ∆PBP5 mutant elevates the ß-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and ß-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.


Assuntos
Alanina/química , Cefalosporinase , Proteínas de Escherichia coli , Ácido Glutâmico/química , Resistência beta-Lactâmica/genética , Alanina/genética , Alanina/metabolismo , Cefalosporinase/química , Cefalosporinase/genética , Cefalosporinase/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Hidrólise , Estrutura Secundária de Proteína/genética
9.
Handb Clin Neurol ; 148: 767-781, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29478613

RESUMO

Obsessive-compulsive disorder (OCD) is present in 1.5-2.5% of the population and can result in substantial lifelong disability. It is characterized by intrusive thoughts, sensations, and urges and by repetitive behaviors that are difficult to control despite, in most cases, preserved insight as to their excessive or irrational nature. The causes and underlying pathophysiology of OCD are not well understood, which has limited the development of new treatments and interventions. Despite evidence for a substantial genetic contribution to disease risk, identification and replication of genetic variants associated with OCD have been challenging. Decades of candidate gene association studies have provided little insight. They are now being supplanted by modern genomewide approaches to discover both common and rare sequence and structural variants. Studies to date suggest potential novel therapeutic avenues such as modulators of glutamatergic and immune pathways; however, individual genetic findings are not yet statistically robust or replicated. Further efforts are clearly needed to identify specific risk variants and to confirm vulnerable pathways by studying much larger cohorts of patients with comprehensive variant discovery approaches. Mouse knockout models have already made notable inroads into our understanding of OCD pathology; their utility will only increase as specific risk alleles are identified.


Assuntos
Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença/genética , Transtorno Obsessivo-Compulsivo/genética , Animais , Modelos Animais de Doenças , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Transdução de Sinais/genética
10.
BMB Rep ; 51(5): 236-241, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29335069

RESUMO

Anoctamin 1 (ANO1) is an anion channel that is activated by changes in cytosolic Ca2+ concentration and noxious heat. Although the critical roles of ANO1 have been elucidated in various cell types, the control of its gating mechanisms by Ca2+ and heat remain more elusive. To investigate critical amino acid residues for modulation of Ca2+ and heat sensing, we constructed a randomized mutant library for ANO1. Among 695 random mutants, reduced Ca2+ sensitivity was observed in two mutants (mutant 84 and 87). Consequently, the E143A mutant showed reduced sensitivity to Ca2+ but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both Ca2+ and noxious heat. These results suggest that the glutamic acids (E) at 143 and 705 residues in ANO1 are critical for modulation of Ca2+ and/or heat responses. Furthermore, these findings help to provide a better understanding of the Ca2+-mediated activation and heat-sensing mechanism of ANO1. [BMB Reports 2018; 51(5): 236-241].


Assuntos
Anoctaminas/química , Anoctaminas/metabolismo , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Temperatura Alta , Nociceptividade , Sequência de Aminoácidos , Animais , Anoctaminas/genética , Ácido Glutâmico/genética , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Modelos Moleculares , Mutação/genética , Domínios Proteicos , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade
11.
Brain Dev ; 40(3): 233-237, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29289389

RESUMO

BACKGROUND: A heterozygous c.1228G > A p.E410K mutation in TUBB3 encoding neuronal-specific ß-tubulin isotype 3 causes TUBB3 E410K syndrome, which exhibits a wide range of neurological and endocrinological abnormalities. CASE DESCRIPTION: The patient is a 31-year-old Japanese woman who was diagnosed with atypical Moebius syndrome because of congenital facial weakness and extraocular ophthalmoplegia sparing abduction. She suffered a femoral neck fracture at 23 years of age, and radiological and endocrinological studies revealed osteoporosis because of hypogonadotropic hypogonadism. She also had borderline intellectual disability, cyclic vomiting, syncope with cough, and decreased sense of smell since childhood. Brain magnetic resonance imaging revealed abnormal morphology of the corpus callosum and pontine. Hypoplastic bilateral oculomotor and facial nerves were evident. Based on these symptoms, we analyzed the TUBB3 gene and identified a heterozygous c.1228G > A (p.E410K) mutation that confirmed the diagnosis of TUBB3 E410K syndrome. CONCLUSION: TUBB3 E410K syndrome may be diagnosed as atypical Moebius syndrome because of overlapping clinical symptoms. Genetic analysis of c.1228G > A in TUBB3 is useful to differentiate TUBB3 E410K syndrome from other disorders presenting congenital external ophthalmoplegia and facial nerve palsy.


Assuntos
Síndrome de Möbius/complicações , Mutação/genética , Osteoporose/genética , Síncope/genética , Tubulina (Proteína)/genética , Adulto , Corpo Caloso/diagnóstico por imagem , Movimentos Oculares/genética , Feminino , Ácido Glutâmico/genética , Humanos , Japão , Lisina/genética , Imagem por Ressonância Magnética , Síndrome de Möbius/diagnóstico por imagem , Osteoporose/complicações , Osteoporose/diagnóstico por imagem , Ponte/diagnóstico por imagem , Síncope/complicações , Síncope/diagnóstico por imagem
12.
Exp Hematol ; 58: 52-58, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28947392

RESUMO

Acute myeloid leukemia (AML) is a blood cancer that is poorly responsive to conventional cytotoxic chemotherapy and a diagnosis of AML is usually fatal. More effective and better-tolerated therapies for AML are desperately needed. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most frequently observed genetic defects in AML. FLT3 inhibitors have shown impressive anti-leukemic activity in clinical trials; however, sustained remissions using these inhibitors as monotherapy have not been achieved. Our previous studies have implicated impaired glutamine metabolism in response to FLT3 inhibitors as a dominant factor causing AML cell death. In this study, we have employed metabolic flux analysis to examine the effects of FLT3 inhibition on glutamine utilization in FLT3-mutated AML cells using stable isotope tracers. We found that the FLT3 inhibitor AC220 inhibited glutamine flux into the antioxidant factor glutathione profoundly due to defective glutamine import. We also found that the glutaminase inhibitor CB-839 similarly impaired glutathione production by effectively blocking flux of glutamine into glutamate. Moreover, the combination of AC220 with CB-839 synergized to deplete glutathione, induce mitochondrial reactive oxygen species, and cause loss of viability through apoptotic cell death. In vivo, glutaminase inhibition with CB-839 facilitated leukemic cell elimination by AC220 and improved survival significantly in a patient-derived xenograft AML mouse model. Therefore, targeting glutaminase in combination with FLT3 may represent an effective therapeutic strategy for improving treatment of FLT3-mutated AML.


Assuntos
Benzenoacetamidas/farmacologia , Benzotiazóis/farmacologia , Glutaminase , Leucemia Mieloide Aguda , Compostos de Fenilureia/farmacologia , Tiadiazóis/farmacologia , Tirosina Quinase 3 Semelhante a fms , Animais , Linhagem Celular Tumoral , Feminino , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
13.
J Gen Virol ; 99(1): 62-72, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29139348

RESUMO

The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I.


Assuntos
Regulação Viral da Expressão Gênica , Ácido Glutâmico/metabolismo , Hepacivirus/metabolismo , Treonina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Ácido Glutâmico/química , Ácido Glutâmico/genética , Células HEK293 , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Mimetismo Molecular , Mutação , Fosforilação , Plasmídeos/química , Plasmídeos/metabolismo , Treonina/química , Treonina/genética , Transfecção , Proteínas não Estruturais Virais/genética
14.
J Alzheimers Dis ; 61(1): 209-219, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29125487

RESUMO

Lipids are considered important factors in the pathogenesis of Alzheimer's disease (AD). In this study, we realized a comparative analysis of the phospholipid profile and phospholipid composition of the temporal cortex from E280A-familiar AD (FAD), sporadic AD (SAD), and healthy human brains. Findings showed a significant decrease of lysophosphatidylcholine and phosphatidylethanolamine formed by low levels of polyunsaturated fatty acids (20 : 4, 22 : 6) in AD brains. However, phosphatidylethanolamine-ceramide and phosphoglycerol were significantly increased in SAD, conformed by high levels of (18 : 0/18 : 1) and (30/32/36 : 0/1/2), respectively. Together, the findings suggest a deficiency in lysophosphacholine and phosphatidylethanolamine, and alteration in the balance between poly- and unsaturated fatty acids in both types of AD, and a differential pattern of phospholipid profile and fatty acid composition between E280A FAD and SAD human brains.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Mutação/genética , Fosfolipídeos/metabolismo , Presenilina-1/genética , Lobo Temporal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alanina/genética , Análise de Variância , Ácidos Graxos/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Ácido Glutâmico/genética , Humanos , Lisofosfatidilcolinas/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Fosfatidiletanolaminas/metabolismo
15.
Mol Biotechnol ; 60(1): 1-11, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29058211

RESUMO

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 °C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lipase/química , Lipase/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Dicroísmo Circular , Estabilidade Enzimática , Ácido Glutâmico/genética , Isoleucina/genética , Lipase/genética , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/genética , Temperatura Ambiente , Valina/genética
16.
Am J Hum Genet ; 101(5): 768-788, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100089

RESUMO

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Deficiência Intelectual/genética , Mutação/genética , Animais , Encéfalo/patologia , Linhagem Celular , Exoma/genética , Feminino , Ácido Glutâmico/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Fosforilação/genética , Transdução de Sinais/genética
17.
Nat Commun ; 8(1): 808, 2017 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-28993643

RESUMO

Males and females use distinct brain circuits to cope with similar challenges. Using RNA sequencing of ribosome-bound mRNA from hippocampal CA3 neurons, we found remarkable sex differences and discovered that female mice displayed greater gene expression activation after acute stress than males. Stress-sensitive BDNF Val66Met mice of both sexes show a pre-stressed translational phenotype in which the same genes that are activated without applied stress are also induced in wild-type mice by an acute stressor. Behaviourally, only heterozygous BDNF Val66Met females exhibit spatial memory impairment, regardless of acute stress. Interestingly, this effect is not observed in ovariectomized heterozygous BDNF Val66Met females, suggesting that circulating ovarian hormones induce cognitive impairment in Met carriers. Cognitive deficits are not observed in males of either genotype. Thus, in a brain region not normally associated with sex differences, this work sheds light on ways that genes, environment and sex interact to affect the transcriptome's response to a stressor.Animals' response to acute stress is known to be influenced by sex and genetics. Here the authors performed RNA-seq on actively translated mRNAs in hippocampal CA3 neurons in mice, and document the effects of sex and genotype (i.e., BDNF Val66Met) on acute stress-induced gene expression.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Biossíntese de Proteínas , Células Piramidais/fisiologia , Estresse Fisiológico/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Regulação da Expressão Gênica , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Masculino , Camundongos Transgênicos , Ovariectomia , RNA Mensageiro , Análise de Sequência de RNA , Fatores Sexuais , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismo
18.
Biochem J ; 474(23): 3871-3886, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29025976

RESUMO

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a ß-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.


Assuntos
Proteínas de Bactérias/química , Ligases/química , Xanthomonas campestris/enzimologia , Acil Coenzima A/química , Acil Coenzima A/genética , Alcenos/química , Alcenos/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ligases/genética , Mutação de Sentido Incorreto , Xanthomonas campestris/genética
19.
Brain ; 140(8): 2210-2225, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28899017

RESUMO

See Huang and Liston (doi:10.1093/awx166) for a scientific commentary on this article.Human depression is associated with glutamatergic dysfunction and alterations in resting state network activity. However, the indirect nature of human in vivo glutamate and activity assessments obscures mechanistic details. Using the chronic social defeat mouse model of depression, we determine how mesoscale glutamatergic networks are altered after chronic stress, and in response to the rapid acting antidepressant, ketamine. Transgenic mice (Ai85) expressing iGluSnFR (a recombinant protein sensor) permitted real-time in vivo selective characterization of extracellular glutamate and longitudinal imaging of mesoscale cortical glutamatergic functional circuits. Mice underwent chronic social defeat or a control condition, while spontaneous cortical activity was longitudinally sampled. After chronic social defeat, we observed network-wide glutamate functional hyperconnectivity in defeated animals, which was confirmed with voltage sensitive dye imaging in an independent cohort. Subanaesthetic ketamine has unique effects in defeated animals. Acutely, subanaesthetic ketamine induces large global cortical glutamate transients in defeated animals, and an elevated subanaesthetic dose resulted in sustained global increase in cortical glutamate. Local cortical inhibition of glutamate transporters in naïve mice given ketamine produced a similar extracellular glutamate phenotype, with both glutamate transients and a dose-dependent accumulation of glutamate. Twenty-four hours after ketamine, normalization of depressive-like behaviour in defeated animals was accompanied by reduced glutamate functional connectivity strength. Altered glutamate functional connectivity in this animal model confirms the central role of glutamate dynamics as well as network-wide changes after chronic stress and in response to ketamine.


Assuntos
Córtex Cerebral/fisiologia , Depressão/fisiopatologia , Ácido Glutâmico/efeitos dos fármacos , Ketamina/farmacologia , Proteínas Vesiculares de Transporte de Glutamato/antagonistas & inibidores , Animais , Antidepressivos/farmacologia , Ácido Aspártico/farmacologia , Comportamento Animal/efeitos dos fármacos , Depressão/metabolismo , Depressão/psicologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Comportamento Social , Imagens com Corantes Sensíveis à Voltagem
20.
J Mol Graph Model ; 77: 153-167, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28858643

RESUMO

Recently, a new signaling complex Death Associated Protein Kinase 1 (DAPK1) ̶ N-methyl-D-aspartate receptor subtype 2B (NMDAR2B or NR2B) engaged in the neuronal death cascade was identified and it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via over-activation of NMDA receptors. An acute brain injury, such as stroke, is a serious life-threatening medical condition which occurs due to poor blood supply to the brain and further leads to neuronal cell death. During a stroke, activated DAPK1 migrates towards the extra-synaptic site and binds to NR2B subunit of NMDA receptor. It is this DAPK1-NR2B interaction that arbitrates the pathological processes like apoptosis, necrosis, and autophagy of neuronal cells observed in stroke injury, hence we aimed to inhibit this vital interaction to prevent neuronal damage. In the present study, using PubChem database, we applied an integrative approach of virtual screening and molecular dynamic simulations and identified a potential lead compound 11 that interrupts DAPK1-NR2B interaction by competing with both ATP and substrate for their binding sites on DAPK1. This inhibitor was found potent and considerably selective to DAPK1 as it made direct contact with the ATP binding sites as well as substrate recognition motifs: Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN). Further in vitro and in vivo experiments are demanded to validate the efficacy of compound 11 nevertheless, it can be considered as suitable starting point for designing DAPK1 inhibitors.


Assuntos
Motivos de Aminoácidos/genética , Proteínas Quinases Associadas com Morte Celular/química , Receptores de N-Metil-D-Aspartato/química , Acidente Vascular Cerebral/tratamento farmacológico , Proteínas Quinases Associadas com Morte Celular/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ácido Glutâmico/química , Ácido Glutâmico/genética , Humanos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ligação Proteica , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
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