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1.
Cell Mol Life Sci ; 77(1): 81-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31728578

RESUMO

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.


Assuntos
Quebras de DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Animais , DNA/química , DNA/genética , DNA Topoisomerases Tipo II/química , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
2.
Chemosphere ; 240: 124902, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31563721

RESUMO

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.


Assuntos
Cádmio/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Aminoaciltransferases , Animais , Disponibilidade Biológica , Cádmio/análise , Ecossistema , Perfilação da Expressão Gênica , Metalotioneína/genética , Metalotioneína/metabolismo , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Estresse Oxidativo/efeitos dos fármacos , Solo , Poluentes do Solo/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
3.
Acta Crystallogr D Struct Biol ; 75(Pt 8): 718-732, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373571

RESUMO

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.


Assuntos
Clostridium perfringens/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Aminoaciltransferases/química , Proteínas de Bactérias/química , Clonagem Molecular/métodos , Cristalografia por Raios X , Cisteína Endopeptidases/química , Escherichia coli/genética , Modelos Moleculares , Polimerização , Domínios Proteicos
4.
Rev Esp Quimioter ; 32 Suppl 3: 3-10, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364335

RESUMO

Ceftobiprole, a novel last generation parenteral cephalosporin, has an extended spectrum of activity, notably against methicillin-resistant Staphylococcus aureus (MRSA), ampicillin-susceptible enterococci, penicillin-resistant pneumococci, Enterobacterales and susceptible Pseudomonas aeruginosa. It exerts an inhibitory action on essential peptidoglycan transpeptidases, interfering with cell wall synthesis. The inhibitory action of ceftobiprole through binding to abnormal PBPs like PBP2a in methicillin-resistant staphylococci and PBP2b and PBP2x in the case of ß-lactam-resistant pneumococci, ultimately leads to rapid bacterial cell death. In the case of Enterobacterales, ceftobiprole retains activity against narrow spectrum ß-lactamases but is hydrolysed by their extended-spectrum counterparts, overexpressed Amp C, and carbapenemases. It is also affected by certain efflux pumps from P. aeruginosa. For anaerobic bacteria, ceftobiprole is active against Gram-positive Clostridioides difficile and Peptococcus spp. and Gram-negative Fusobacterium nucleatum but not against Bacteroides group or other anaerobic Gram-negatives. In in vitro studies, a low propensity to select for resistant subpopulations has been demonstrated. Currently, ceftobiprole is approved for the treatment of community-acquired pneumonia and hospital-acquired pneumonia with the exception of ventilator-associated pneumonia. Ceftobiprole's place in therapy appears to lie mainly in its combined activity against Gram-positive organisms, such as S. aureus and S. pneumoniae alongside that against Gram-negative organisms such as P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/metabolismo , Cefalosporinas/metabolismo , Infecções Comunitárias Adquiridas/tratamento farmacológico , Endopeptidases/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Enterococcus/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Resistência às Penicilinas , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia
5.
ACS Chem Biol ; 14(8): 1836-1844, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31348637

RESUMO

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.


Assuntos
Corantes Fluorescentes/química , Vírus da Influenza A/fisiologia , Proteínas de Membrana/metabolismo , Peptídeos/química , Rodaminas/química , Aminoaciltransferases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Cães , Endossomos/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Internalização do Vírus
6.
Adv Mater ; 31(33): e1902462, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31265196

RESUMO

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Cisteína Endopeptidases/química , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/química , Proteínas Recombinantes/química , Aminoaciltransferases/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Cisteína Endopeptidases/administração & dosagem , Dissulfetos/química , Liberação Controlada de Fármacos , Humanos , Luz , Oxirredução , Peptídeos/química , Fotólise , Polietilenoglicóis/química , Proteínas Recombinantes/administração & dosagem , Staphylococcus aureus/enzimologia
7.
J Plant Physiol ; 240: 153011, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357099

RESUMO

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.


Assuntos
Aminoaciltransferases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Tabaco/fisiologia , Aminoaciltransferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relação Dose-Resposta a Droga , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Tabaco/genética
8.
Int J Med Microbiol ; 309(5): 359-363, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31182276

RESUMO

Daptomycin has become an important antibiotic for the treatment of serious Methicillin-Resistant Staphylococcus aureus (MRSA) infections. Unlike other approved antibiotics, its mode of action is still under active investigation, as well as the molecular basis of daptomycin resistance, which emerges in some cases during daptomycin treatment. Small nucleotide polymorphisms (SNPs) in the Multiple Peptide Resistance Factor (MprF) appear to play a major role in the resistance mechanism. Until recently, the impact of the SNPs on MprF activity has remained unclear, which is due to conflicting reports on resistance-associated phenotypes and an incomplete understanding of the mode of action of MprF. However, recent structural insights into MprF and studies with isogenic mutants have now led to a new model of MprF-mediated daptomycin resistance, which harmonizes most of the observed phenotypes and provides a basis for challenging biochemical investigations.


Assuntos
Aminoaciltransferases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Farmacorresistência Bacteriana/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Aminoaciltransferases/química , Proteínas de Bactérias/química , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Mutação , Polimorfismo de Nucleotídeo Único , Infecções Estafilocócicas/tratamento farmacológico
9.
Mycorrhiza ; 29(4): 389-395, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31218402

RESUMO

Arbuscular mycorrhizal fungi (AMF) are considered a potential biotechnological tool for mitigating heavy metal (HM) toxicity. A greenhouse experiment was conducted to evaluate the impacts of the AM fungus Rhizophagus irregularis on cadmium (Cd) uptake, mycorrhizal colonization, and some plant growth parameters of Medicago sativa (alfalfa) in Cd-polluted soils. In addition, expression of two metal chelators (MsPCS1 (phytochelatin synthase) and MsMT2 (metallothionein)) and two metal transporter genes (MsIRT1 and MsNramp1) was analyzed using quantitative real-time PCR (qRT-PCR). Cd addition had a significant negative effect on mycorrhizal colonization. However, AMF symbiosis promoted the accumulation of biomass under both stressed and unstressed conditions compared with non-mycorrhizal (NM) plants. Results also showed that inoculation with R. irregularis significantly reduced shoot Cd concentration in polluted soils. Transcripts abundance of MsPCS1, MsMT2, MsIRT1, and MsNRAMP1 genes were downregulated compared with NM plants indicating that metal sequestration within hyphal fungi probably made Cd concentration insufficient in root cells for induction of these genes. These results suggest that reduction of shoot Cd concentration in M. sativa colonized by R. irregularis could be a promising strategy for safe production of this plant in Cd-polluted soils.


Assuntos
Aminoaciltransferases/genética , Cádmio/metabolismo , Glomeromycota/fisiologia , Medicago sativa/metabolismo , Metalotioneína/genética , Micorrizas/fisiologia , Proteínas de Plantas/genética , Poluentes do Solo/metabolismo , Aminoaciltransferases/metabolismo , Transporte Biológico , Medicago sativa/genética , Medicago sativa/microbiologia , Metalotioneína/metabolismo , Proteínas de Plantas/metabolismo , Simbiose
10.
Ecotoxicol Environ Saf ; 180: 295-308, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31100594

RESUMO

Crops can become contaminated when grown in soils containing heavy metals. Cadmium is a heavy metal that poses a significant health risk to humans. The purpose of this study was to evaluate the effect of cadmium on lettuce (Lactuca sativa Linn) and the contamination risk of lettuce grown in cadmium environments. The results showed that photosynthesis and growth parameters were significantly affected by cadmium. Lettuce has the ability to absorb large amounts of cadmium from the contaminated environment and so is a cadmium hyperaccumulator plant. The study showed that approximately 35% of the total absorbed cadmium is transmitted to aerial and edible parts of lettuce. This study was undertaken as lettuce has the ability to absorb and accumulate high levels of cadmium. There are however are no reports on the PCS gene and the potential for high cadmium accumulation in lettuce. The bioinformatics study revealed that lettuce has two phytochelatin synthase genes that produce 6 PCSs through splicing leading to the ability of lettuce to store high levels of cadmium. These six sequences although different in length have high similarity. Sequence structure, cellular location, three-dimensional structure, phylogeny and a comparison of their catalytic power were evaluated. The high accumulation of cadmium in lettuce and the presence of several PCSs contribute to the accumulation of cadmium in aerial tissues. The cultivation of lettuce in contaminated environments led us to evaluate suspected farms for the presence of cadmium in produce. Lettuce grown in industrial environments contaminated with cadmium can pose a serious threat to human health.


Assuntos
Aminoaciltransferases/genética , Cádmio/toxicidade , Alface/efeitos dos fármacos , Poluentes do Solo/toxicidade , Solo/química , Cádmio/análise , Cádmio/metabolismo , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/enzimologia , Alface/enzimologia , Fotossíntese/efeitos dos fármacos , Poluentes do Solo/análise , Poluentes do Solo/metabolismo , Inquéritos e Questionários
11.
PLoS One ; 14(5): e0217369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125361

RESUMO

Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6-8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the ß-cyclodextrin (ß-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized ß-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Química Click/métodos , Reação de Cicloadição/métodos , Ciclodextrinas/química , Cisteína Endopeptidases/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Dendrímeros/síntese química , Dendrímeros/química , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Biossíntese Peptídica , Peptídeos/síntese química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Chemosphere ; 230: 488-497, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31121512

RESUMO

Azolla is a floating aquatic fern, having amazing capacity for concentrating toxic heavy metals. Metallothioneins (MTs) and phytochelatins (PCs) are well-defined heavy metal-binding ligands in plants. Bioaccumulation potential of different Azolla species varies according to their heavy metal ions. Therefore, the accumulation of Ni, Zn, Cu, and Cd was studied in A. pinnata, A. filiculoides, and a sample taken from Anzali wetland. Moreover, the expression of metallothionein and phytochelatin synthase encoding genes was examined at different metal concentrations. The highest level of Cu and Cd absorption was detected in A. pinnata, while the maximum amount of Ni and Zn absorption was observed in A. filiculoides and the sample taken from Anzali, respectively. The MT2 and PCS1 gene expression patterns were significantly induced by the heavy metal treatments, confirming their roles in phytoremediation potential of Azolla. However, as the results concerning heavy metal accumulation and gene expression vary in different species, only specific species of Azolla can be used for special purposes. It can be concluded that the Azolla is a good candidate for phytoremediation purposes, and the formation of phytochelatin-heavy metal complexes and their sequestration in vacuole are the main processes influencing susceptibility of Azolla to heavy metals.


Assuntos
Aminoaciltransferases/genética , Gleiquênias/metabolismo , Expressão Gênica/efeitos dos fármacos , Metalotioneína/genética , Metais Pesados/análise , Poluentes Químicos da Água/análise , Aminoaciltransferases/metabolismo , Organismos Aquáticos/genética , Organismos Aquáticos/metabolismo , Biodegradação Ambiental , Gleiquênias/genética , Metalotioneína/metabolismo , Metais Pesados/metabolismo , Poluentes Químicos da Água/metabolismo
13.
Anim Genet ; 50(3): 279-282, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974000

RESUMO

Glutaminyl-peptide cyclotransferase-like (QPCTL) is an isoenzyme of glutaminyl-peptide cyclotransferase (QPCT). QPCTL and QPCT catalyze the formation of N-terminal modified pyroglutamate-fractalkine and the chemokine CCL2. The objective of this study was to investigate the association between insertions/deletions in the chicken QPCTL promoter region with growth traits in chickens. We first detected two insertion/deletion variants of QPCTL via whole-genome resequencing analysis of DNA samples from Xichuan chickens. A total of 1896 individuals from 12 breeds were genotyped for 52- and 224-bp insertions/deletions. We found two novel insertions/deletions in the promoter region of the chicken QPCTL gene and studied their association with chicken body weight and carcass traits. Our findings show that QPCTL can be a molecular marker for chicken genetics and breeding programs.


Assuntos
Aminoaciltransferases/genética , Galinhas/genética , Mutação INDEL , Regiões Promotoras Genéticas , Animais , Proteínas Aviárias/genética , Galinhas/classificação
14.
Plant Biol (Stuttg) ; 21(5): 854-861, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30929297

RESUMO

Cadmium (Cd) is one of the most toxic heavy metals and a non-essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised. To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl2 using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15). The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector-transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/-15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/-15 resulted in toxicity due to excess phytochelatin production in A. thaliana. These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.


Assuntos
Aminoaciltransferases/metabolismo , Cádmio/toxicidade , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Aminoaciltransferases/genética , Arabidopsis , Genes de Plantas/genética , Oryza/efeitos dos fármacos , Oryza/genética , Plantas Geneticamente Modificadas , Alinhamento de Sequência
15.
J Microbiol ; 57(6): 431-443, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30900148

RESUMO

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.


Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/classificação , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Cisteína Endopeptidases/química , Cisteína Endopeptidases/classificação , Actinobacteria/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/fisiologia , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Simulação por Computador , Cisteína/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/fisiologia , Fímbrias Bacterianas , Genoma Bacteriano , Proteínas de Membrana , Modelos Moleculares , Filogenia , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Análise de Sequência
16.
Nat Med ; 25(4): 612-619, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833751

RESUMO

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells2-5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.


Assuntos
Aminoaciltransferases/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Receptores Imunológicos/metabolismo , Aminoaciltransferases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos Transgênicos , Neoplasias/patologia , Proteínas Opsonizantes/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo
17.
Microbiol Spectr ; 7(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30737913

RESUMO

Sortases cleave short peptide motif sequences at the C-terminal end of secreted surface protein precursors and either attach these polypeptides to the peptidoglycan of Gram-positive bacteria or promote their assembly into pilus structures that are also attached to peptidoglycan. Sortase A, the enzyme first identified in the human pathogen Staphylococcus aureus, binds LPXTG motif sorting signals, cleaves between threonine (T) and glycine (G) residues, and forms an acyl enzyme between its active-site cysteine thiol and the carboxyl group of threonine (T). Sortase A acyl enzyme is relieved by the nucleophilic attack of the cross bridge amino group within lipid II, thereby generating surface protein linked to peptidoglycan precursor. Such products are subsequently incorporated into the cell wall envelope by enzymes of the peptidoglycan synthesis pathway. Surface proteins linked to peptidoglycan may be released from the bacterial envelope to diffuse into host tissues and fulfill specific biological functions. S. aureus sortase A is essential for host colonization and for the pathogenesis of invasive diseases. Staphylococcal sortase-anchored surface proteins fulfill key functions during the infectious process, and vaccine-induced antibodies targeting surface proteins may provide protection against S. aureus. Alternatively, small-molecule inhibitors of sortase may be useful agents for the prevention of S. aureus colonization and invasive disease.


Assuntos
Aminoaciltransferases/imunologia , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Peptidoglicano/metabolismo , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Anticorpos Antibacterianos/imunologia , Humanos , Infecções Estafilocócicas/patologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia
18.
Infect Immun ; 87(5)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804098

RESUMO

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis.


Assuntos
Aminoaciltransferases/fisiologia , Proteínas de Bactérias/fisiologia , Parede Celular/efeitos dos fármacos , Cisteína Endopeptidases/fisiologia , Enterococcus/fisiologia , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/fisiologia , Animais , Modelos Animais de Doenças , Trato Gastrointestinal/microbiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
19.
Bioorg Med Chem ; 27(5): 721-728, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711310

RESUMO

New compounds able to counteract staphylococcal biofilm formation are needed. In this study we investigate the mechanism of action of pyrrolomycins, whose potential as antimicrobial agents has been demonstrated. We performed a new efficient and easy method to use microwave organic synthesis suitable for obtaining pyrrolomycins in good yields and in suitable amount for their in vitro in-depth investigation. We evaluate the inhibitory activity towards Sortase A (SrtA), a transpeptidase responsible for covalent anchoring in Gram-positive peptidoglycan of many surface proteins involved in adhesion and in biofilm formation. All compounds show a good inhibitory activity toward SrtA, having IC50 values ranging from 130 to 300 µM comparable to berberine hydrochloride. Of note compound 1d shows a good affinity in docking experiment to SrtA and exhibits the highest capability to interfere with biofilm formation of S. aureus showing an IC50 of 3.4 nM. This compound is also effective in altering S. aureus murein hydrolase activity that is known to be responsible for degradation, turnover, and maturation of bacterial peptidoglycan and involved in the initial stages of S. aureus biofilm formation.


Assuntos
Antibacterianos/farmacologia , Pirróis/farmacologia , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana , Micro-Ondas , Simulação de Acoplamento Molecular , N-Acetil-Muramil-L-Alanina Amidase/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pirróis/síntese química , Pirróis/farmacocinética , Staphylococcus aureus/efeitos dos fármacos
20.
Curr Med Chem ; 26(18): 3260-3278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30678614

RESUMO

Benzimidazole scaffold has been efficiently used for the design of various pharmacologically active molecules. Indeed, there are various benzimidazole drugs, available today, employed for the treatment of different diseases. Although there is no benzimidazole moiety containing a drug used in clinic today for the treatment of Alzheimer's Disease (AD), there have been many benzimidazole derivative compounds designed and synthesized to act on some of the validated and non-validated targets of AD. This paper aims to review the literature to describe these benzimidazole containing molecules designed to target some of the biochemical cascades shown to be involved in the development of AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Benzimidazóis/química , Inibidores da Colinesterase , Aminoaciltransferases/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Descoberta de Drogas , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos/química , Humanos
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