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1.
Nucleic Acids Res ; 52(5): 2498-2518, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38300764

RESUMO

The SGI1 family integrative mobilizable elements, which are efficient agents in distribution of multidrug resistance in Gammaproteobacteria, have a complex, parasitic relationship with their IncC conjugative helper plasmids. Besides exploiting the transfer apparatus, SGI1 also hijacks IncC plasmid control mechanisms to time its own excision, replication and expression of self-encoded T4SS components, which provides advantages for SGI1 over its helpers in conjugal transfer and stable maintenance. Furthermore, SGI1 destabilizes its helpers in an unknown, replication-dependent way when they are concomitantly present in the same host. Here we report how SGI1 exploits the helper plasmid partitioning system to displace the plasmid and simultaneously increase its own stability. We show that SGI1 carries two copies of sequences mimicking the parS sites of IncC plasmids. These parS-like elements bind the ParB protein encoded by the plasmid and increase SGI1 stability by utilizing the parABS system of the plasmid for its own partitioning, through which SGI1 also destabilizes the helper plasmid. Furthermore, SGI1 expresses a small protein, Sci, which significantly strengthens this plasmid-destabilizing effect, as well as SGI1 maintenance. The plasmid-induced replication of SGI1 results in an increased copy-number of parS-like sequences and Sci expression leading to strong incompatibility with the helper plasmid.


Assuntos
Elementos de DNA Transponíveis , Salmonella , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Salmonella/efeitos dos fármacos , Salmonella/genética , Farmacorresistência Bacteriana Múltipla
2.
Nucleic Acids Res ; 49(2): 832-846, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406256

RESUMO

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDCSGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriVSGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter PS004. This replicon transiently maintains SGI1 as a 4-8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.


Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Conjugação Genética/genética , Farmacorresistência Bacteriana Múltipla/genética , Sequências Repetitivas Dispersas/genética , Salmonella typhimurium/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica/genética , Genes Reporter , Integrases/metabolismo , Óperon/genética , Filogenia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Recombinases/metabolismo , Replicon/genética , Alinhamento de Sequência , Transativadores/genética , Transativadores/metabolismo
3.
J Biol Chem ; 296: 100255, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33837736

RESUMO

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.


Assuntos
Antígenos de Superfície/ultraestrutura , Ligação Proteica/genética , Receptores de Antígenos de Linfócitos T/ultraestrutura , Linfócitos T/ultraestrutura , Antígenos de Superfície/química , Antígenos de Superfície/genética , Humanos , Complexo Principal de Histocompatibilidade/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/ultraestrutura , Linfócitos T/química , Linfócitos T/imunologia , Timócitos/química , Timócitos/ultraestrutura
4.
Mol Microbiol ; 116(6): 1533-1551, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34784078

RESUMO

The SGI1-family elements that are specifically mobilized by the IncA- and IncC-family plasmids are important vehicles of antibiotic resistance among enteric bacteria. Although SGI1 exploits many plasmid-derived conjugation and regulatory functions, the basic mobilization module of the island is unrelated to that of IncC plasmids. This module contains the oriT and encodes the mobilization proteins MpsA and MpsB, which belong to the tyrosine recombinases and not to relaxases. Here we report an additional, essential transfer factor of SGI1. This is a small RNA deriving from the 3'-end of a primary RNA that can also serve as mRNA of ORF S022. The functional domain of this sRNA named sgm-sRNA is encoded between the mpsA gene and the oriT of SGI1. Terminator-like sequence near the promoter of the primary transcript possibly has a regulatory function in controlling the amount of full-length primary RNA, which is converted to the active sgm-sRNA through consecutive maturation steps influenced by the 5'-end of the primary RNA. The mobilization module of SGI1 seems unique due to its atypical relaxase and the newly identified sgm-sRNA, which is required for the horizontal transfer of the island but appears to act differently from classical regulatory sRNAs.


Assuntos
Transferência Genética Horizontal , Ilhas Genômicas , RNA Bacteriano/genética , Salmonella/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , Farmacorresistência Bacteriana Múltipla , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , Salmonella/metabolismo
5.
J Biomol NMR ; 68(1): 19-32, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28434103

RESUMO

Pseudocontact shifts (PCS) encode long-range information on 3D structures of protein backbones and side-chains. The level of structural detail that can be obtained increases with the number of different sites tagged with a paramagnetic metal ion to generate PCSs. Here we show that PCSs from two different sites can suffice to determine the structure of polypeptide chains and their location and orientation relative to the magnetic susceptibility tensor χ, provided that PCSs are available for 1H as well as heteronuclear spins. In addition, PCSs from two different sites are shown to provide detailed structural information on the conformation of methyl group-bearing amino-acid side-chains. A previously published ensemble structure of ubiquitin is shown to explain the magnetic susceptibility and alignment tensors slightly better than structures that try to explain the experimental data by a single conformation, illustrating the potential of PCSs as a tool to investigate small conformational changes.


Assuntos
Elementos da Série dos Lantanídeos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Ubiquitina/química , Aminoácidos de Cadeia Ramificada/química , Conformação Proteica , Proteínas/química
6.
Bioconjug Chem ; 28(6): 1741-1748, 2017 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-28485576

RESUMO

Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.


Assuntos
DNA/química , Elementos da Série dos Lantanídeos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Sítios de Ligação , Oligonucleotídeos Fosforotioatos/química
7.
Bioorg Med Chem Lett ; 27(2): 254-260, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27923620

RESUMO

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.


Assuntos
Corantes Fluorescentes/química , Inflamação/metabolismo , Organofosfonatos/química , Serina Proteases/análise , Animais , Colite/metabolismo , Corantes Fluorescentes/síntese química , Camundongos , Estrutura Molecular , Organofosfonatos/síntese química , Pancreatite/metabolismo , Serina Proteases/metabolismo
8.
Nucleic Acids Res ; 43(18): 8735-45, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26209134

RESUMO

The genomic island SGI1 and its variants, the important vehicles of multi-resistance in Salmonella strains, are integrative elements mobilized exclusively by the conjugative IncA/C plasmids. Integration and excision of the island are carried out by the SGI1-encoded site-specific recombinase Int and the recombination directionality factor Xis. Chromosomal integration ensures the stable maintenance and vertical transmission of SGI1, while excision is the initial step of horizontal transfer, followed by conjugation and integration into the recipient. We report here that SGI1 not only exploits the conjugal apparatus of the IncA/C plasmids but also utilizes the regulatory mechanisms of the conjugation system for the exact timing and activation of excision to ensure efficient horizontal transfer. This study demonstrates that the FlhDC-family activator AcaCD, which regulates the conjugation machinery of the IncA/C plasmids, serves as a signal of helper entry through binding to SGI1 xis promoter and activating SGI1 excision. Promoters of int and xis genes have been identified and the binding site of the activator has been located by footprinting and deletion analyses. We prove that expression of xis is activator-dependent while int is constitutively expressed, and this regulatory mechanism is presumably responsible for the efficient transfer and stable maintenance of SGI1.


Assuntos
Conjugação Genética , Ilhas Genômicas , Plasmídeos/genética , Salmonella/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Genes Bacterianos , Regiões Promotoras Genéticas , Transativadores/metabolismo
9.
Orv Hetil ; 158(5): 163-171, 2017 Feb.
Artigo em Húngaro | MEDLINE | ID: mdl-28132538

RESUMO

The association of diabetes and heart failure is very common, furthermore, the pathophysiology and clinical course of the two entities have many crossing-points. Today, the spectrum of available anti-diabetic drugs is extremely wide, ranging from the classical (insulin, biguanides, sulphonylureas) to the most recent agents (gliptins, gliflozins). The cardiovascular effects of these drugs are multiple, their knowledge is important in the everyday practice, as the use of safe drugs regarding of heart failure is preferred. Our work provides an overview of each class of drugs after the presentation of the mechanism of action and the main representatives, the effects on the cardiovascular system, including those on heart failure will be described, mentioning the results of the most important clinical trials. The available data confirm the beneficial effects of metformin and gliflozins and the harmful effect of thiazolidinediones in heart failure. The other classes of drugs are permitted in heart failure, but it is important to continuously monitor the signs of decompensation. Orv. Hetil., 2017. 158(5), 163-171.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Tiazolidinedionas/efeitos adversos , Tiazolidinedionas/uso terapêutico , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Quimioterapia Combinada , Insuficiência Cardíaca/prevenção & controle , Humanos , Hipoglicemiantes/efeitos adversos , Metformina/efeitos adversos
10.
Antimicrob Agents Chemother ; 60(11): 6780-6786, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27600047

RESUMO

Two A/C incompatibility group (IncA/C family) plasmids from the 1960s have been sequenced and classified into the A/C2 type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 genomic island not previously found in the family. In the 173.1-kb R16a, the 29.9-kb antibiotic resistance island (ARI) is located in a unique backbone position not utilized by ARIs. ARIR16a consists of Tn1, Tn6020, and Tn6333, harboring the resistance genes blaTEM-1D and aphA1b and a mer module, respectively; a truncated Tn5393 copy; and a gene cluster with unknown function. Plasmid IP40a is 170.4 kb in size and contains a 5.6-kb ARI inserted into the kfrA gene. ARIIP40a carrying blaTEM-1D and aphA1b genes is composed of Tn1 with a Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186, and Tn1000 insertions scattered in the backbone; an IS150 copy in GIsul2; and a complete Tn6333 carrying a mer module at the position of ARIR16a Loss of resistance markers in R16a, IP40a, and R55 was observed during stability tests. Every phenotypic change proved to be the result of recombination events involving mobile elements. Intramolecular transposition of IS copies that generated IP40a derivatives lacking large parts of the backbone could account for the formation of other family members, too. The MinION platform proved to be a valuable tool in bacterial genome sequencing since it generates long reads that span repetitive elements and facilitates full-length plasmid or chromosome assembly. Nanopore technology enables rapid characterization of large, low-copy-number plasmids and their rearrangement products.


Assuntos
DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Genoma Bacteriano , Plasmídeos/química , Plasmídeos/história , beta-Lactamases/genética , Antibacterianos/farmacologia , Automação Laboratorial , Conjugação Genética , Elementos de DNA Transponíveis , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Ilhas Genômicas , História do Século XX , Plasmídeos/metabolismo , Análise de Sequência de DNA
11.
J Biomol NMR ; 66(4): 281-293, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27988858

RESUMO

Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca2+-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.


Assuntos
Elementos da Série dos Lantanídeos/química , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/química , Neurônios , Ressonância Magnética Nuclear Biomolecular , Proteínas SNARE/química , Animais , Marcação por Isótopo , Espectroscopia de Ressonância Magnética/métodos , Complexos Multiproteicos/metabolismo , Neurônios/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ratos , Proteínas SNARE/metabolismo
12.
Bioorg Med Chem ; 21(13): 3760-79, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23673221

RESUMO

Novel N-hydroxyalkyl-2-aminophenothiazines implying a tetrazole moiety at the alkyl chain have been synthesized by hydroboration-oxidation of dienes followed by Buchwald-Hartwig cross-coupling reaction. Also, some sulfoxide and sulfone derivatives have been prepared by selective oxidations. MDR inhibition studies on rat hepatocyte cell culture revealed that some derivatives exhibit marked biological efficacy exceeding that of the standard verapamil (e.g., 3h, 4h, 16). Selected derivatives were subjected to chemical resolution to provide both enantiomers which were shown of similar activity on P-gp interaction measurements. The new compounds exhibited no toxicity.


Assuntos
Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenotiazinas/química , Fenotiazinas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aminação , Animais , Células Cultivadas , Masculino , Fenotiazinas/síntese química , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/química , Sulfonas/farmacologia , Sulfóxidos/síntese química , Sulfóxidos/química , Sulfóxidos/farmacologia
13.
Int J Med Microbiol ; 302(1): 4-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22000740

RESUMO

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential.


Assuntos
Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/microbiologia , Plasmídeos/genética , Doenças dos Suínos/microbiologia , Resistência a Tetraciclina/genética , Fatores de Virulência/genética , Animais , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Loci Gênicos , Humanos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Plasmídeos/isolamento & purificação , Análise de Sequência de DNA , Suínos , Tetraciclina/farmacologia , Virulência
14.
PLoS One ; 17(7): e0271414, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35901099

RESUMO

The E. coli element IS30 has adopted the copy-out-paste-in transposition mechanism that is prevalent in a number of IS-families. As an initial step, IS30 forms free circular transposition intermediates like IS minicircles or tandem IS-dimers by joining the inverted repeats of a single element or two, sometimes distantly positioned IS copies, respectively. Then, the active IR-IR junction of these intermediates reacts with the target DNA, which generates insertions, deletions, inversions or cointegrates. The element shows dual target specificity as it can insert into hot spot sequences or next to its inverted repeats. In this study the pathways of rearrangements of transposition-derived cointegrate-like structures were examined. The results showed that the probability of further rearrangements in these structures depends on whether the IS elements are flanked by hot spot sequences or take part in an IR-IR junction. The variability of the deriving products increases with the number of simultaneously available IRs and IR-IR joints in the cointegrates or the chromosome. Under certain conditions, the parental structures whose transposition formed the cointegrates are restored and persist among the rearranged products. Based on these findings, a novel dynamic model has been proposed for IS30, which possibly fits to other elements that have adopted the same transposition mechanism. The model integrates the known transposition pathways and the downstream rearrangements occurring after the formation of different cointegrate-like structures into a complex network. Important feature of this network is the presence of "feedback loops" and reversible transposition rearrangements that can explain how IS30 generates variability and preserves the original genetic constitution in the bacterial population, which contributes to the adaptability and evolution of host bacteria.


Assuntos
Elementos de DNA Transponíveis , Escherichia coli , Sequência de Bases , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Humanos , Plasmídeos
15.
Magn Reson (Gott) ; 3(1): 1-13, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37905175

RESUMO

The metallo-ß-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

16.
Bioorg Med Chem Lett ; 21(22): 6782-7, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21982493

RESUMO

We have synthesised an extensive series of URB602 analogues as inhibitors of monoacylglycerol lipase (MAGL), which is the major enzyme responsible for metabolising the endocannabinoid 2-arachidonylglycerol. The recently identified crystal structure of MAGL was used in the design strategy and revealed three possible binding sites for URB602 and the proposed analogues. A test series of carbamate analogues were docked into the identified sites to predict the most favourable binding location. The synthesised analogues of URB602 explored the biological effects of isosteric replacement, ring size and substitution, para substitution of the biphenyl moiety and the incorporation of a bicyclic element. The compounds were tested for their ability to inhibit human MAGL. The carbamate analogue 16 displayed the most significant inhibitory activity, reducing MAGL activity to 26% of controls at 100 µM compared to 73% for the parent compound URB602.


Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Compostos de Bifenilo/síntese química , Domínio Catalítico , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Humanos , Modelos Moleculares , Monoacilglicerol Lipases/química , Ligação Proteica , Relação Estrutura-Atividade
17.
J Bacteriol ; 192(13): 3414-23, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20418401

RESUMO

The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested.


Assuntos
Sequências Repetidas Terminais/genética , Sítios de Ligação/genética , Elementos de DNA Transponíveis , Ensaio de Desvio de Mobilidade Eletroforética , Mutação , Plasmídeos/genética
18.
Front Microbiol ; 11: 539, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32318036

RESUMO

INTRODUCTION: The emergence and spread of new strains of zoonotic bacteria, such as multidrug resistant (MDR) Salmonella Infantis, represent a growing health risk for humans in and outside Europe due to foodborne infections of poultry meat origin. OBJECTIVES: In order to understand genome relations of S. Infantis strains from Hungary and from different geographic regions, we performed a comprehensive genome analysis of nine Hungarian and 67 globally selected strains of S. Infantis and 26 Salmonella strains representing 13 non-Infantis serovars. RESULTS: Analyses of whole-, and accessory genomes, showed that almost all S. Infantis strains were separated from the non-Infantis serovars. S. Infantis strains from Hungary formed subclusters based on their time of isolation. In whole genome sequence analysis, the Swiss strains of S. Infantis were closely related to each other and clustered together with subclusters of strains from Hungary, Japan, Italy, United States, and Israel. The accessory genome analysis revealed that the Swiss strains were distinct from most of the strains investigated, including the Hungarian ones. Analysis of the cloud genes offered the most detailed insight into the genetic distance and relationship of S. Infantis strains confirming that the Swiss and Hungarian strains belonged to different lineages. As expected, core genome analysis provided the least discriminatory power for analysis of S. Infantis. Genomic sequences of nine strains from Brazil, Israel, Mexico, Nigeria, and Senegal (deposited as S. Infantis) proved to be outliers from the S. Infantis clade. They were predicted to be Salmonella Rissen, Salmonella Ouakarm, Salmonella Kentucky, Salmonella Thompson, and Salmonella enterica subsp. diarizonae. CONCLUSION: Accessory genome of S. Infantis showed the highest diversity suggesting a faster evolution than that of the whole genomes contributing to the emergence of multiple genetic variants of S. Infantis worldwide. Accordingly, in spite of the comprehensive analysis of several genomic characteristics, no epidemiologic links between these S. Infantis strains from different countries could be established. It is also concluded that several strains originally designated as S. Infantis need in databanks reclassification.

19.
Nat Neurosci ; 23(5): 625-637, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284608

RESUMO

Decades of research support the idea that associations between a conditioned stimulus (CS) and an unconditioned stimulus (US) are encoded in the lateral amygdala (LA) during fear learning. However, direct proof for the sources of CS and US information is lacking. Definitive evidence of the LA as the primary site for cue association is also missing. Here, we show that calretinin (Calr)-expressing neurons of the lateral thalamus (Calr+LT neurons) convey the association of fast CS (tone) and US (foot shock) signals upstream from the LA in mice. Calr+LT input shapes a short-latency sensory-evoked activation pattern of the amygdala via both feedforward excitation and inhibition. Optogenetic silencing of Calr+LT input to the LA prevents auditory fear conditioning. Notably, fear conditioning drives plasticity in Calr+LT neurons, which is required for appropriate cue and contextual fear memory retrieval. Collectively, our results demonstrate that Calr+LT neurons provide integrated CS-US representations to the LA that support the formation of aversive memories.


Assuntos
Condicionamento Clássico/fisiologia , Medo/fisiologia , Vias Neurais/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Complexo Nuclear Basolateral da Amígdala/fisiologia , Calreticulina/metabolismo , Sinais (Psicologia) , Memória/fisiologia , Camundongos , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Tálamo/fisiologia
20.
ACS Chem Biol ; 15(3): 718-727, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32022538

RESUMO

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.


Assuntos
Catepsinas/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes/química , Aminoácidos/química , Animais , Técnicas de Cultura de Células , Linhagem Celular , Diazometano/química , Humanos , Hidrocarbonetos Fluorados/química , Cetonas/química , Rim/citologia , Rim/diagnóstico por imagem , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Domínios Proteicos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Especificidade por Substrato
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