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1.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1640-1649, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924362

RESUMO

Ethyl carbamate (EC) is a carcinogen detected in fermented foods and alcohol beverages. Excessive intake of EC is possibly harmful to health. Enzymatic degradation is one of the most effective approaches for reducing EC in fermented foods. Urease catalyzes the hydrolysis of both EC and urea. This confers urease a good application prospect in reducing EC and its precursor urea in fermented foods. Currently, degradation of EC in alcohol beverages by urease is inefficient due to its low urethanase activity and poor affinity to EC. Urease from Bacillus amyloliquefaciens JP-21 was successfully expressed in Escherichia coli at the level of 3 292 U/L urease and 227.3 U/L urethanase. Two key residues M326 and M374 were characterized that might block the binding of enzyme to EC, through simulating docking the structure of catalytic subunit UreC of urease with EC. Three mutants (M374A, M374T and M326V) of urease with improved urethanase activity were obtained by performing point saturated mutagenesis approach. Using EC as the substrate, Km values of M374A, M374T and M326V were detected to be 101.8 mmol/L, 129.5 mmol/L and 121.7 mmol/L, respectively, which were decreased by 37.47%-50.82% compared with that of the wild type urease. These mutants can degrade more than 97% of urea in rice wine and mutant M374T shows the highest degradation of EC in rice wine. EC content in rice wine was reduced from 525 µg/L to 393 µg/L by using M374T, and the EC degradation rate of it is 0.97 folds higher than that of the wild type urease. The results are of great significance for engineering the catalytic properties of urease and improving its industrial properties, and lays a good foundation for developing strategies to reducing microbial metabolic ammonia (amine) hazards in fermented foods.


Assuntos
Bacillus amyloliquefaciens , Microbiologia de Alimentos , Oryza , Urease , Uretana , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Carcinógenos/metabolismo , Mutagênese Sítio-Dirigida , Urease/genética , Urease/metabolismo , Uretana/metabolismo , Vinho/microbiologia
2.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
3.
Nat Commun ; 11(1): 4160, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814772

RESUMO

Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin's activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation.


Assuntos
Grelina/metabolismo , Fenilalanina/metabolismo , Receptores de Grelina/metabolismo , Animais , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Grelina/química , Grelina/genética , Células HEK293 , Humanos , Ligantes , Camundongos Endogâmicos MRL lpr , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fenilalanina/genética , Ligação Proteica , Conformação Proteica , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/genética , Células Sf9 , Spodoptera
4.
Proc Natl Acad Sci U S A ; 117(34): 20520-20529, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32796105

RESUMO

As paradigms for proton-coupled electron transfer in enzymes and benchmarks for a fully renewable H2 technology, [FeFe]-hydrogenases behave as highly reversible electrocatalysts when immobilized on an electrode, operating in both catalytic directions with minimal overpotential requirement. Using the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1) we have conducted site-directed mutagenesis and protein film electrochemistry to determine how efficient catalysis depends on the long-range coupling of electron and proton transfer steps. Importantly, the electron and proton transfer pathways in [FeFe]-hydrogenases are well separated from each other in space. Variants with conservative substitutions (glutamate to aspartate) in either of two positions in the proton-transfer pathway retain significant activity and reveal the consequences of slowing down proton transfer for both catalytic directions over a wide range of pH and potential values. Proton reduction in the variants is impaired mainly by limiting the turnover rate, which drops sharply as the pH is raised, showing that proton capture from bulk solvent becomes critical. In contrast, hydrogen oxidation is affected in two ways: by limiting the turnover rate and by a large overpotential requirement that increases as the pH is raised, consistent with the accumulation of a reduced and protonated intermediate. A unique observation having fundamental significance is made under conditions where the variants still retain sufficient catalytic activity in both directions: An inflection appears as the catalytic current switches direction at the 2H+/H2 thermodynamic potential, clearly signaling a departure from electrocatalytic reversibility as electron and proton transfers begin to be decoupled.


Assuntos
Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Chlamydomonas reinhardtii , Clostridium , Transporte de Elétrons , Hidrogenase/genética , Proteínas com Ferro-Enxofre/genética , Mutagênese Sítio-Dirigida , Prótons
5.
Proc Natl Acad Sci U S A ; 117(33): 20298-20304, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747539

RESUMO

In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ∼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of ∼1,900 Å3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating.


Assuntos
Domínios Proteicos , Dobramento de Proteína , Canais de Cátion TRPM/química , Animais , Temperatura Baixa , Microscopia Crioeletrônica , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oócitos , Conformação Proteica , Canais de Cátion TRPM/metabolismo , Termodinâmica , Xenopus laevis
6.
PLoS One ; 15(6): e0235341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603354

RESUMO

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.


Assuntos
Aldeído Liases/química , Aldeído Liases/genética , Domínio Catalítico , Cristalografia por Raios X/métodos , Esterases/química , Esterases/genética , Hevea/genética , Hevea/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida/métodos , Proteínas de Plantas/genética , Dobramento de Proteína , Especificidade por Substrato , Tabaco/genética , Tabaco/metabolismo
7.
PLoS One ; 15(7): e0235925, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639967

RESUMO

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.


Assuntos
Síndrome de Angelman/genética , Ubiquitina-Proteína Ligases/metabolismo , Síndrome de Angelman/patologia , Biocatálise , Dicroísmo Circular , Humanos , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
8.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
9.
Nat Commun ; 11(1): 3146, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561718

RESUMO

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Biossíntese de Proteínas/fisiologia , Capuzes de RNA/metabolismo , Biologia Computacional , Fator de Iniciação 4E em Eucariotos/genética , Proteínas Intrinsicamente Desordenadas/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/fisiologia
10.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1232-1240, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597073

RESUMO

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.


Assuntos
Vetores Genéticos , Mutagênese Sítio-Dirigida , Técnicas de Amplificação de Ácido Nucleico , Sequência de Bases , Clonagem Molecular , Vetores Genéticos/genética , Mutagênese Sítio-Dirigida/métodos , Plasmídeos , Reação em Cadeia da Polimerase
11.
PLoS Genet ; 16(6): e1008848, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32530919

RESUMO

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Flagelos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação , Polissacarídeos Bacterianos/biossíntese , Pseudomonas aeruginosa/patogenicidade , Seleção Genética
12.
Arch Biochem Biophys ; 689: 108436, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32492375

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium (Kv) and cyclic nucleotide-gated (CNG) channels. HCN channels contain the glycine-tyrosine-glycine (GYG) sequence that forms part of the selectivity filter, a similar structure than some potassium channels; however, they permeate both sodium and potassium, giving rise to an inward current. Yet a second amino acid sequence, leucine-cysteine-isoleucine (LCI), next to GYG, is well-preserved in all HCNs but not in the selective potassium channels. In this study we used site-directed mutagenesis and electrophysiology in frog oocytes to determine whether the LCI sequence affects the kinetics of HCN2 currents. Permeability and voltage dependence were evaluated, and we found a role of LCI in the gating mechanism combined with changes in ion permeability. The I residue resulted critical to this function.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Ativação do Canal Iônico , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Permeabilidade , Potássio/metabolismo , Sódio/metabolismo , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
13.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510506

RESUMO

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.


Assuntos
Culex/embriologia , Culex/genética , Edição de Genes , Microinjeções/métodos , Mosquitos Vetores/genética , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/patogenicidade , Animais , Culex/virologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/virologia , Feminino , Mutagênese Sítio-Dirigida , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologia
14.
Proc Natl Acad Sci U S A ; 117(27): 15573-15580, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571944

RESUMO

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.


Assuntos
Proteínas de Bactérias/metabolismo , Evolução Molecular , Luz , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Cor , Cianobactérias/genética , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Mutagênese Sítio-Dirigida , Nostoc/genética , Nostoc/metabolismo , Nostoc/efeitos da radiação , Fotobiologia/métodos , Fotorreceptores Microbianos/efeitos da radiação , Biologia Sintética/métodos , Tetrapirróis/metabolismo
15.
Eur J Med Chem ; 200: 112410, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32492596

RESUMO

CXCR4, a well-studied coreceptor of human immunodeficiency virus type 1 (HIV-1) entry, recognizes its cognate ligand SDF-1α (also named CXCL12) which plays many important roles, including regulating immune cells, controlling hematopoietic stem cells, and directing cancer cells migration. These pleiotropic roles make CXCR4 an attractive target to mitigate human disorders. Here a new class of symmetrical polyamines was designed and synthesized as potential small molecule CXCR4 antagonists. Among them, a representative compound 21 (namely HF50731) showed strong CXCR4 binding affinity (mean IC50 = 19.8 nM) in the CXCR4 competitive binding assay. Furthermore, compound 21 significantly inhibited SDF-1α-induced calcium mobilization and cell migration, and blocked HIV-1 infection via antagonizing CXCR4 coreceptor function. The structure-activity relationship analysis, site-directed mutagenesis, and molecular docking were conducted to further elucidate the binding mode of compound 21, suggesting that compound 21 could primarily occupy the minor subpocket of CXCR4 and partially bind in the major subpocket by interacting with residues W94, D97, D171, and E288. Our studies provide not only new insights for the fragment-based design of small molecule CXCR4 antagonists for clinical applications, but also a new and effective molecular probe for CXCR4-targeting biological studies.


Assuntos
Desenho de Fármacos , Poliaminas/síntese química , Receptores CXCR4/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Quimiocina CXCL12 , Infecções por HIV/prevenção & controle , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Poliaminas/farmacologia
16.
Proc Natl Acad Sci U S A ; 117(25): 14512-14521, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513714

RESUMO

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Domínios Proteicos/genética , Regulação Alostérica , Animais , Cátions Bivalentes/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/ultraestrutura , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos , Técnicas de Patch-Clamp , Prolina/genética , Conformação Proteica em alfa-Hélice/genética , Relação Estrutura-Atividade , Xenopus laevis
17.
PLoS One ; 15(6): e0231513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479540

RESUMO

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of ß-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of ß-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular ß-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing ß-1,2-linked arabinofuranose disaccharide (ß-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) ß-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the ß-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 ß-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. ß-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the ß-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.


Assuntos
Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Bifidobacterium longum/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Alinhamento de Sequência
18.
PLoS One ; 15(5): e0232090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357153

RESUMO

Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocyanins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we isolated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydroquercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydromyricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns.


Assuntos
Oxirredutases do Álcool/genética , Antocianinas/biossíntese , Dryopteris/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Catálise , Dryopteris/enzimologia , Dryopteris/metabolismo , Mutagênese Sítio-Dirigida , Filogenia , Alinhamento de Sequência , Especificidade por Substrato
19.
Nat Commun ; 11(1): 2365, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398758

RESUMO

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.


Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Transglutaminases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Domínio Catalítico/genética , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Transglutaminases/genética , Transglutaminases/isolamento & purificação , Transglutaminases/ultraestrutura , Ubiquitina/isolamento & purificação , Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitinação
20.
PLoS One ; 15(5): e0233503, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32442184

RESUMO

Recently-emerged base editing technologies could create single base mutations at precise genomic positions without generation DNA double strand breaks. Herbicide resistant mutations have been successfully introduced to different plant species, including Arabidopsis, watermelon, wheat, potato and tomato via C to T (or G to A on the complementary strand) base editors (CBE) at the P197 position of endogenous acetolactate synthase (ALS) genes. Additionally, G to A conversion to another conserved amino acid S653 on ALS gene could confer tolerance to imidazolinone herbicides. However, no such mutation was successfully generated via CBE, likely due to the target C base is outside of the classic base editing window. Since CBE driven by egg cell (EC) specific promoter would re-edit the wild type alleles in egg cells and early embryos, we hypothesized the diversity of base editing outcomes could be largely increased at later generations to allow selection of desired herbicide resistant mutants. To test this hypothesis, we aimed to introduce C to T conversion to the complement strand of S653 codon at ALS gene, hosting a C at the 10th position within the 20-nt spacer sequence outside of the classic base editing window. While we did not detect base-edited T1 plants, efficient and diverse base edits emerged at later generations. Herbicide resistant mutants with different editing outcomes were recovered when T3 and T4 seeds were subject to herbicide selection. As expected, most herbicide resistant plants contained S653N mutation as a result of G10 to A10. Our results showed that CBE could create imidazolinone herbicide resistant trait in Arabidopsis and be potentially applied to crops to facilitate weed control.


Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Resistência a Herbicidas/genética , Acetolactato Sintase/genética , Substituição de Aminoácidos , Proteínas de Arabidopsis/genética , Sequência de Bases , Sistemas CRISPR-Cas , DNA de Plantas/genética , Edição de Genes , Genes de Plantas , Herbicidas/farmacologia , Imidazolinas/farmacologia , Mutagênese Sítio-Dirigida , Melhoramento Vegetal , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Seleção Genética , Controle de Plantas Daninhas
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