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1.
Nucleic Acids Res ; 47(18): 9777-9788, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504788

RESUMO

The homochirality of amino acids is vital for the functioning of the translation apparatus. l-Amino acids predominate in proteins and d-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in d-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNA-synthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of d-amino acids recognition. Here, we showed that d-alanine was effectively activated by AlaRS and d-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that d-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes d-aminoacyl-tRNAs, was inactive against d-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.


Assuntos
Alanina-tRNA Ligase/genética , RNA de Transferência/genética , Thermus thermophilus/enzimologia , Tirosina-tRNA Ligase/genética , Alanina/genética , Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Aminoacilação/genética , Escherichia coli/genética , Hidrólise
2.
J Zhejiang Univ Sci B ; 20(8): 660-669, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31273963

RESUMO

Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/metabolismo , Listeriose/microbiologia , Fator sigma/metabolismo , Alanina/genética , Bacillus subtilis , Sítios de Ligação , Deleção de Genes , Teste de Complementação Genética , Homeostase , Concentração de Íons de Hidrogênio , Mutação , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Estresse Fisiológico
3.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269680

RESUMO

Much of the experimental data, especially in life sciences, is considered to be useless if it demonstrates a large standard deviation from the mean value. The Renaissance distribution, as presented in this study, allows one to extract true values from such statistical data with large noise. To obtain proof of the Renaissance distribution, high-throughput synthesis of deep substitutions for a target amino acid sequence was performed, and the known epitope was identified in assay with human serum antibodies. In addition, the Renaissance distribution was shown to approach the epitope affinity maturation by the deep alanine substitution. The Renaissance distribution may have an impact in the development of novel specific drugs.


Assuntos
Alanina/genética , Substituição de Aminoácidos , Peptídeos/genética , Alanina/química , Sequência de Aminoácidos , Epitopos/química , Epitopos/genética , Humanos , Modelos Moleculares , Peptídeos/química , Análise Serial de Proteínas , Distribuições Estatísticas , Processos Estocásticos
4.
Endocrinology ; 160(5): 1333-1347, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951171

RESUMO

The transcription factor forkhead box O1 (FoxO1) is a key mediator in the insulin signaling pathway and controls multiple physiological functions, including hepatic glucose production (HGP) and pancreatic ß-cell function. We previously demonstrated that S256 in human FOXO1 (FOXO1-S256), equivalent to S253 in mouse FoxO1 (FoxO1-S253), is a key phosphorylation site mediating the effect of insulin as a target of protein kinase B on suppression of FOXO1 activity and expression of target genes responsible for gluconeogenesis. Here, we investigated the role of FoxO1-S253 phosphorylation in control of glucose homeostasis in vivo by generating global FoxO1-S253A/A knockin mice, in which FoxO1-S253 alleles were replaced with alanine (A substitution) blocking FoxO1-S253 phosphorylation. FoxO1-S253A/A mice displayed mild increases in feeding blood glucose and insulin levels but decreases in fasting blood glucose and glucagon concentrations, as well as a reduction in the ratio of pancreatic α-cells/ß-cells per islet. FoxO1-S253A/A mice exhibited a slight increase in energy expenditure but barely altered food intake and glucose uptake among tissues. Further analyses revealed that FoxO1-S253A/A enhances FoxO1 nuclear localization and promotes the effect of glucagon on HGP. We conclude that dephosphorylation of S253 in FoxO1 may reflect a molecular basis of pancreatic plasticity during the development of insulin resistance.


Assuntos
Proteína Forkhead Box O1/metabolismo , Glucose/metabolismo , Homeostase , Serina/metabolismo , Alanina/genética , Alanina/metabolismo , Animais , Glicemia/metabolismo , Proteína Forkhead Box O1/genética , Glucagon/sangue , Insulina/sangue , Resistência à Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Serina/genética
5.
Nucleic Acids Res ; 47(9): 4707-4720, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916338

RESUMO

Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.


Assuntos
Proteínas Mutantes/genética , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Alanina/genética , Sequência de Aminoácidos/genética , Dimerização , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Ácido Glutâmico/genética , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Mutação , Ribonuclease III/química , Ribonuclease III/isolamento & purificação , Análise de Sequência de DNA , Especificidade por Substrato
6.
J Biol Chem ; 294(14): 5677-5687, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30737278

RESUMO

Membrane fusion is the first essential step in HIV-1 replication. The gp41 subunit of HIV-1 envelope protein (Env), a class I fusion protein, achieves membrane fusion by forming a structure called a six-helix bundle composed of N- and C-terminal heptad repeats. We have recently shown that the distal portion of the α9 helix in the C-terminal heptad repeat of X4-tropic HXB2 Env plays a critical role in the late-stage membrane fusion and viral infection. Here, we used R5-tropic JRFL Env and constructed six alanine insertion mutants, 641+A to 646+A, in the further distal portion of α9 where several glutamine residues are conserved (the number corresponds to the position of the inserted alanine in JRFL Env). 644+A showed the most severe defect in syncytia formation. Decreased fusion pore formation activity, revealed by a dual split protein assay, was observed in all mutants except 641+A. Sequence analysis and substitution of inserted 644A with Gln revealed that the glutamine residue at position 644 that forms complex hydrogen-bond networks with other polar residues on the surface of the six-helix bundle is critical for cell-cell fusion. We also developed a split NanoLuc® (Nluc) reporter-based assay specific to the virus-cell membrane fusion step to analyze several of the mutants. Interestingly syncytia-competent mutants failed to display Nluc activities. In addition to defective fusion activity, a reduction of Env incorporation into virions may further contribute to differences in cell-cell and virus-cell fusions.


Assuntos
Bioensaio , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Fusão de Membrana , Mutagênese Insercional , Internalização do Vírus , Alanina/genética , Alanina/metabolismo , Linhagem Celular , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Humanos , Estrutura Secundária de Proteína
7.
Biotechnol J ; 14(7): e1800643, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30791213

RESUMO

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.


Assuntos
Alanina/genética , Proteínas Fúngicas/genética , Glicosídeo Hidrolases , Penicillium , Fatores de Transcrição/genética , Arabinose/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Engenharia Genética , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/metabolismo , Mutação/genética , Mutação/fisiologia , Penicillium/genética , Penicillium/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
8.
J Biol Chem ; 294(2): 716-725, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30425098

RESUMO

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 µm, versus 9.5 µm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 µm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 µm, observed with P106L alone, to 10.3 µm The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Substituição de Aminoácidos , Glicina/análogos & derivados , Herbicidas/metabolismo , Zea mays/enzimologia , Zea mays/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/química , Agrobacterium/enzimologia , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Glicina/química , Glicina/genética , Glicina/metabolismo , Mutagênese , Zea mays/efeitos dos fármacos , Zea mays/metabolismo
9.
Biochemistry ; 58(3): 166-170, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30406995

RESUMO

Methyltransferases (MTases) are superfamilies of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM), a nucleoside-based cofactor, to a wide variety of substrates such as DNA, RNA, proteins, small molecules, and lipids. Depending upon their structural features, the MTases can be further classified into different classes; we consider exclusively the largest class of MTases, the Rossmann-fold MTases. It has been shown that the nucleoside cofactor-binding Rossmann enzymes, particularly the nicotinamide adenine dinucleotide (NAD)-, flavin adenine dinucleotide (FAD)-, and SAM-binding MTases enzymes, share common binding motifs that include a Gly-rich loop region that interacts with the cofactor and a highly conserved acidic residue (Asp/Glu) that interacts with the ribose moiety of the cofactor. Here, we observe that the Gly-rich loop region of the Rossmann MTases adapts a specific type II' ß-turn in the proximity of the cofactor (<4 Å), and it appears to be a key feature of these superfamilies. Additionally, we demonstrate that the conservation of this ß-turn could play a critical role in the enzyme-cofactor interaction, thereby shedding new light on the structural conformation of the Gly-rich loop region from Rossmann MTases.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Alanina/genética , Sítios de Ligação , Coenzimas/química , Coenzimas/metabolismo , Simulação por Computador , Flavina-Adenina Dinucleotídeo/química , Glicina/genética , Glicina/metabolismo , Metilação , Metiltransferases/genética , Mutagênese , NAD/metabolismo , Conformação Proteica , Dobramento de Proteína , S-Adenosilmetionina/química
10.
G3 (Bethesda) ; 9(1): 145-151, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413416

RESUMO

Mud1 is an inessential 298-amino acid protein subunit of the Saccharomyces cerevisiae U1 snRNP. Mud1 consists of N-terminal and C-terminal RRM domains (RRM1 and RRM2) separated by a linker domain. Synthetic lethal interactions of mud1∆ with deletions of inessential spliceosome components Nam8, Mud2, and Msl1, or missense mutations in the branchpoint-binding protein Msl5 enabled us to dissect genetically the domain requirements for Mud1 function. We find that the biological activities of Mud1 can be complemented by co-expressing separately the RRM1 (aa 1-127) and linker-RRM2 (aa 128-298) modules. Whereas RRM1 and RRM2 (aa 197-298) per se are inactive in all tests of functional complementation, the linker-RRM2 by itself partially complements a subset of synthetic lethal mud1∆ interactions. Linker segment aa 155 to 196 contains a nuclear localization signal rich in basic amino acids that is necessary for RRM2 activity in mud1∆ complementation. Alanine scanning mutagenesis indicates that none of the individual RRM1 amino acid contacts to U1 snRNA in the cryo-EM model of the yeast U1 snRNP is necessary for mud1∆ complementation activity.


Assuntos
Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/genética , Alanina/genética , Sequência de Aminoácidos , Epistasia Genética/genética , Domínios Proteicos/genética , Processamento de RNA/genética , Fatores de Processamento de RNA/genética , Saccharomyces cerevisiae/genética , Deleção de Sequência/genética , Spliceossomos/genética , Fator de Processamento U2AF/genética , Mutações Sintéticas Letais/genética
11.
Mar Drugs ; 16(12)2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30551685

RESUMO

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure⁻activity relationship (SAR) between GI and mouse α1ß1δε nAChRs. The Pro5, Gly8, Arg8, and Tyr11 were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1ß1δε nAChR. On the contrary, substituting Asn4, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1ß1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1ß1δε nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse α1ß1δε nAChR in detail that will help to develop the novel analogues of GI.


Assuntos
Alanina/genética , Conotoxinas/química , Caramujo Conus , Antagonistas Nicotínicos/química , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos/genética , Animais , Conotoxinas/farmacologia , Conotoxinas/uso terapêutico , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Músculo Esquelético/metabolismo , Mutagênese , Doenças Neuromusculares/tratamento farmacológico , Junção Neuromuscular/metabolismo , Antagonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/uso terapêutico , Oócitos , Técnicas de Patch-Clamp , Receptores Nicotínicos/química , Relação Estrutura-Atividade , Transmissão Sináptica/efeitos dos fármacos , Xenopus laevis
12.
Sci Signal ; 11(560)2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538174

RESUMO

Increased abundance of GRK2 [G protein-coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.


Assuntos
Apoptose , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Glucose/química , Insuficiência Cardíaca/prevenção & controle , Isquemia/fisiopatologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Animais , Quinase 2 de Receptor Acoplado a Proteína G/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Camundongos , Mitocôndrias/patologia , Miócitos Cardíacos/patologia , Oxirredução , Consumo de Oxigênio , Fosforilação , Mutação Puntual , Serina/química , Serina/genética , Serina/metabolismo , Transdução de Sinais
13.
PLoS One ; 13(11): e0207526, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30462715

RESUMO

Follicle-stimulating hormone receptor (FSHR) is a G-protein coupled receptor (GPCR) and a prototype of the glycoprotein hormone receptors subfamily of GPCRs. Structural data of the FSHR ectodomain in complex with follicle-stimulating hormone suggests a "pull and lift" activation mechanism that triggers a conformational change on the seven α-helix transmembrane domain (TMD). To analyze the conformational changes of the FSHR TMD resulting from sequence variants associated with reproductive impairment in humans, we set up a computational approach combining helix modeling and molecular simulation methods to generate conformational ensembles of the receptor at room (300 K) and physiological (310 K) temperatures. We examined the receptor dynamics in an explicit membrane environment of polyunsaturated phospholipids and solvent water molecules. The analysis of the conformational dynamics of the functional (N680 and S680) and dysfunctional (mutations at D408) variants of the FSHR allowed us to validate the FSHR-TMD model. Functional variants display a concerted motion of flexible intracellular regions at TMD helices 5 and 6. Disruption of side chain interactions and conformational dynamics were detected upon mutation at D408 when replaced with alanine, arginine, or tyrosine. Dynamical network analysis confirmed that TMD helices 2 and 5 may share communication pathways in the functional FSHR variants, whereas no connectivity was detected in the dysfunctional mutants, indicating that the global dynamics of the FSHR was sensitive to mutations at amino acid residue 408, a key position apparently linked to misfolding and variable cell surface plasma membrane expression of FSHRs with distinct mutations at this position.


Assuntos
Aminoácidos/química , Hormônio Foliculoestimulante/química , Conformação Molecular , Receptores do FSH/química , Alanina/química , Alanina/genética , Sequência de Aminoácidos/genética , Aminoácidos/genética , Simulação por Computador , Hormônio Foliculoestimulante/genética , Humanos , Bicamadas Lipídicas/química , Lipídeos/química , Lipídeos/genética , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Receptores do FSH/genética , Transdução de Sinais
14.
Mov Disord ; 33(11): 1734-1739, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30288781

RESUMO

BACKGROUND: The p.A53T point mutation in the α-synuclein gene (SNCA) is a rare but highly relevant cause of autosomal dominant Parkinson's disease (PD). OBJECTIVES: The objective of this study was to assess striatal dopaminergic denervation in a cohort of symptomatic carriers of the p.A53T SNCA mutation as compared to PD patients. METHODS: Data from the Parkinson's Progression Markers Initiative database of 11 symptomatic p.A53T SNCA mutation carriers who underwent 123I-FP-CIT SPECT [(123) I-2ß-carbomethoxy-3ß-(4-iodophenyl)-N-(3-fluoropropyl) nortropane single photon emission computed tomography] imaging at our site were compared with those of 33 age-, sex-, and disease duration-matched PD patients. RESULTS: The p.A53T mutation carriers had significantly lower caudate nucleus binding ratio both contralaterally and ipsilaterally to the most affected side (P = .002 and P = .006) and a decreased contralateral caudate/putamen signal ratio (P = .007) as compared to PD. A similar degree of striatal asymmetry was observed in both subgroups. No correlation between scores in neuropsychological tests and caudate nucleus dopaminergic denervation could be demonstrated. CONCLUSIONS: PD patients harboring the p.A53T SNCA mutation show evidence of a more severe nigrostriatal denervation, especially evident in the caudate nucleus. The lack of significant differences in the putaminal binding ratios may reflect a floor effect or a true preferential targeting of the caudate terminals in p.A53T SNCA-associated PD. © 2018 International Parkinson and Movement Disorder Society.


Assuntos
Mutação/genética , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/genética , Tomografia Computadorizada de Emissão de Fóton Único , Tropanos , alfa-Sinucleína/genética , Adulto , Alanina/genética , Transtornos Cognitivos/diagnóstico por imagem , Transtornos Cognitivos/etiologia , Estudos de Coortes , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Feminino , Lateralidade Funcional , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/complicações , Treonina/genética , Tropanos/farmacocinética
15.
J Biol Rhythms ; 33(5): 497-514, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30175684

RESUMO

Within the suprachiasmatic nucleus (SCN)-the locus of the master circadian clock- transcriptional regulation via the CREB/CRE pathway is implicated in the functioning of the molecular clock timing process, and is a key conduit through which photic input entrains the oscillator. One event driving CRE-mediated transcription is the phosphorylation of CREB at serine 133 (Ser133). Indeed, numerous reporter gene assays have shown that an alanine point mutation in Ser133 reduces CREB-mediated transcription. Here, we sought to examine the contribution of Ser133 phosphorylation to the functional role of CREB in SCN clock physiology in vivo. To this end, we used a CREB knock-in mouse strain, in which Ser133 was mutated to alanine (S/A CREB). Under a standard 12 h light-dark cycle, S/A CREB mice exhibited a marked alteration in clock-regulated wheel running activity. Relative to WT mice, S/A CREB mice had highly fragmented bouts of locomotor activity during the night phase, elevated daytime activity, and a delayed phase angle of entrainment. Further, under free-running conditions, S/A CREB mice had a significantly longer tau than WT mice and reduced activity amplitude. In S/A CREB mice, light-evoked clock entrainment, using both Aschoff type 1 and 6 h "jet lag" paradigms, was markedly reduced relative to WT mice. S/A CREB mice exhibited attenuated transcriptional drive, as assessed by examining both clock-gated and light-evoked gene expression. Finally, SCN slice culture imaging detected a marked disruption in cellular clock phase synchrony following a phase-resetting stimulus in S/A CREB mice. Together, these data indicate that signaling through CREB phosphorylation at Ser133 is critical for the functional fidelity of both SCN timing and entrainment.


Assuntos
Relógios Circadianos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Serina/metabolismo , Núcleo Supraquiasmático/fisiologia , Alanina/genética , Animais , Ritmo Circadiano , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Técnicas de Introdução de Genes , Camundongos , Atividade Motora , Proteínas Circadianas Period/genética , Fosforilação , Serina/genética , Proteínas tau/metabolismo
16.
J Biol Chem ; 293(46): 17985-17996, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30237166

RESUMO

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.


Assuntos
Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/biossíntese , Ácidos Teicoicos/metabolismo , Tioléster Hidrolases/metabolismo , Alanina/genética , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases/metabolismo , Proteínas de Transporte/metabolismo , Ensaios Enzimáticos , Histidina/química , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Serina/química , Staphylococcus aureus/enzimologia , Tioléster Hidrolases/química , Tioléster Hidrolases/genética
17.
ACS Chem Biol ; 13(10): 2855-2861, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30216041

RESUMO

Improvements in genetic code expansion have made preparing proteins with diverse functional groups almost routine. Nonetheless, unnatural amino acids (Uaas) pose theoretical burdens on protein solubility, and determinants of position-specific tolerability to Uaas remain underexplored. To broadly examine associations, we systematically assessed the effect of substituting the fluorescent Uaa, acridonylalanine, at more than 50 chemically, evolutionarily, and structurally diverse residues in two bacterial proteins: LexA and RecA. Surprisingly, properties that ostensibly contribute to Uaa tolerability-such as conservation, hydrophobicity, or accessibility-demonstrated no consistent correlations with resulting protein solubility. Instead, solubility is closely dependent on the location of the substitution within the overall tertiary structure, suggesting that intrinsic properties of protein domains, and not individual positions, are stronger determinants of Uaa tolerability. Consequently, those who seek to install Uaas in new target proteins should consider broadening, rather than narrowing, the types of residues screened for Uaa incorporation.


Assuntos
Acridonas/química , Alanina/análogos & derivados , Alanina/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Corantes Fluorescentes/química , Serina Endopeptidases/química , Serina Endopeptidases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutagênese , Engenharia de Proteínas/métodos , Solubilidade
18.
EMBO Rep ; 19(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30150323

RESUMO

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.


Assuntos
Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Proteases Específicas de Ubiquitina/genética , Alanina/genética , Substituição de Aminoácidos/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Catálise , Cisteína/genética , Enzimas Desubiquitinantes/química , Endopeptidases/química , Humanos , Mutação/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transativadores/química , Ubiquitina/química , Ubiquitina/genética , Proteases Específicas de Ubiquitina/química , Ubiquitinação/genética
19.
Artigo em Inglês | MEDLINE | ID: mdl-30150481

RESUMO

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; consequently, extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (MAb) that targets AT and is currently in phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function. To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2- to 27-fold) for 7 out of 9 mutants, and no binding was detected for the W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants.


Assuntos
Alanina/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/genética , Mutagênese/genética , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Células A549 , Animais , Antibacterianos/farmacologia , Anticorpos Monoclonais Humanizados , Epitopos/genética , Epitopos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia
20.
Biochem Biophys Res Commun ; 503(4): 2619-2624, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30100068

RESUMO

Intracellular trafficking is a tightly regulated cellular process, mediated in part by Rab GTPases and their corresponding effector proteins. Viruses have evolved mechanisms to hijack these processes to promote their lifecycles. Here we describe a mechanism by which cleavage of the Rab7 adaptor protein, RILP (Rab interacting lysosomal protein) is induced by viral infection. We report that RILP is directly cleaved by caspase-1 and we have identified a novel caspase-1 recognition site at aspartic acid 75 within the RILP sequence. Alanine substitution at D75 blocks caspase-1-mediated RILP cleavage. Full-length RILP localizes in a tight vesicular structure near the perinuclear region while the cleaved form of RILP re-distributes throughout the cytoplasm. However, cleavage alone was insufficient to re-localize RILP to the cellular periphery and re-localization required specific phosphorylation events near the caspase-1 recognition site. The combination of cleavage and phosphorylation were both needed for release from the dynein component p150Glued and redistribution of CD63+ve intracellular vesicles.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Caspase 1/genética , Complexo Dinactina/genética , Tetraspanina 30/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Transporte Biológico , Caspase 1/metabolismo , Vesículas Citoplasmáticas/química , Complexo Dinactina/metabolismo , Dineínas/genética , Dineínas/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Mutação , Fosforilação , Proteólise , Transdução de Sinais , Tetraspanina 30/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
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