RESUMO
BACKGROUND: HIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps. RESULTS: Here we report that C-A1 inhibits HIV-1 integration in a capsid-dependent way. Using molecular docking, we identify an extended binding pocket delimited by two adjacent capsid monomers where C-A1 is predicted to bind. Isothermal titration calorimetry confirmed that C-A1 binds to hexameric capsid. Cyclosporine washout assays in Jurkat CD4+ T cells expressing engineered human TRIMCyp showed that C-A1 causes faster and greater escape from TRIMCyp restriction. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D capsid mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintain some integrity of the viral core in the nucleus. Furthermore C-A1 increased binding of CPSF6, a nuclear protein, to capsid. CONCLUSIONS: Our results indicate that capsid is involved in post-nuclear entry steps preceding integration.
Assuntos
Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/fisiologia , Internalização do Vírus , Aminocumarinas/metabolismo , Antivirais/metabolismo , Linhagem Celular , HIV-1/efeitos dos fármacos , HumanosRESUMO
Vicinal dithiol-containing proteins (VDPs) play a significant role in maintaining the cellular redox homeostasis and are implicated in many diseases. To provide new chemical tools for VDPs imaging, we report here a ratiometric fluorescent probe CAsH2 for VDPs using 7-diethylaminiocoumarin as the fluorescent reporter and cyclic 1,3,2-dithiarsenolane as the specific ligand. CAsH2 shows peculiar dual fluorescence emission from the excited intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) states in aqueous media. However, upon selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, causing the excited state ICT to TICT conversion to be restricted; as a result, an increase from the ICT emission band and a decrease from the TICT emission band were observed simultaneously. The designed probe shows high selectivity toward VDPs over other proteins and biological thiols. Preliminary experiments show that CAsH2 can be used for the ratiometric imaging of endogenous VDPs in living cells. So far as we know, this is a rare example of the ratiometric fluorescent probe designed via modulating the ICT-TICT conversion process, which provides a new way to construct various protein-specific ratiometric fluorescent probes.
Assuntos
Aminocumarinas/química , Arsenicais/química , Corantes Fluorescentes/química , Proteínas/análise , Compostos de Sulfidrila/análise , Aminocumarinas/metabolismo , Aminocumarinas/efeitos da radiação , Animais , Arsenicais/metabolismo , Arsenicais/efeitos da radiação , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/efeitos da radiação , Humanos , Luz , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Proteínas/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Aminocoumarins are potent antibiotics belonging to a relatively small group of secondary metabolites produced by actinomycetes. Genome mining of Catenulispora acidiphila has recently led to the discovery of a gene cluster responsible for biosynthesis of novel aminocoumarins, cacibiocins. However, regulation of the expression of this novel gene cluster has not yet been analyzed. In this study, we identify transcriptional regulators of the cacibiocin gene cluster. Using a heterologous expression system, we show that the CabA and CabR proteins encoded by cabA and cabR genes in the cacibiocin gene cluster control the expression of genes involved in the biosynthesis, modification, regulation, and potentially, efflux/resistance of cacibiocins. CabA positively regulates the expression of cabH (the first gene in the cabHIYJKL operon) and cabhal genes encoding key enzymes responsible for the biosynthesis and halogenation of the aminocoumarin moiety, respectively. We provide evidence that CabA is a direct inducer of cacibiocin production, whereas the second transcriptional factor, CabR, is involved in the negative regulation of its own gene and cabT-the latter of which encodes a putative cacibiocin transporter. We also demonstrate that CabR activity is negatively regulated in vitro by aminocoumarin compounds, suggesting the existence of analogous regulation in vivo. Finally, we propose a model of multilevel regulation of gene transcription in the cacibiocin gene cluster by CabA and CabR.
Assuntos
Actinomycetales/genética , Aminocumarinas/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Genes Bacterianos , Genoma Bacteriano , Fatores de Transcrição/genética , Actinomycetales/química , Actinomycetales/metabolismo , Sequência de Aminoácidos , Aminocumarinas/química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Óperon , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L(-1) in C. acidiphila to 60 mg L(-1) in S. coelicolor M1152. By HR-MS and NMR analysis, cacibiocin A was found to contain a 3-amino-4,7-dihydroxycoumarin moiety linked by an amide bond to a pyrrole-2,5-dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6' and 8' of the aminocoumarin moiety.
Assuntos
Actinomycetales/química , Aminocumarinas/química , Antibacterianos/química , Pirróis/química , Actinomycetales/genética , Actinomycetales/metabolismo , Aminocumarinas/metabolismo , Aminocumarinas/farmacologia , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Genoma Bacteriano , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Conformação Molecular , Família Multigênica , Pirróis/metabolismo , Pirróis/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Streptomyces/metabolismoRESUMO
Coumermycin A1 is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It exhibits potent antibacterial and anticancer activity. The coumermycin A1 molecule contains two terminal 5-methyl-pyrrole-2-carboxylic acid moieties and one central 3-methylpyrrole-2,4-dicarboxylic acid moiety (CPM). While the biosynthesis of the terminal moieties has been elucidated in detail, the pathway leading to the CPM remains poorly understood. In this work, the minimal set of genes required for the generation of the CPM scaffold was identified. It comprises the five genes couR1, couR2a, couR2b, couR3, and couR4 which are grouped together in a contiguous 4.7 kb region within the coumermycin A1 biosynthetic gene cluster. The DNA fragment containing these genes was cloned into an expression plasmid and heterologously expressed in Streptomyces coelicolor M1146. Thereupon, the formation of CPM could be shown by HPLC and by HPLC-MS/MS, in comparison to an authentic CPM standard. This proves that the genes couR1-couR4 are sufficient to direct the biosynthesis of CPM, and that the adjacent genes couR5 and couR6 are not required for this pathway. The enzyme CouR3 was expressed in Escherichia coli and purified to near homogeneity. The protein exhibited an ATPase activity similar to that reported for its close ortholog, the threonine kinase PduX. However, we could not show a threonine kinase activity of CouR3, and; therefore, the substrate of CouR3 in CPM biosynthesis is still unknown and may be different from threonine.
Assuntos
Aminocumarinas/metabolismo , Antibacterianos/metabolismo , Ácidos Dicarboxílicos/metabolismo , Pirróis/metabolismo , Streptomyces/genética , Adenosina Trifosfatases/metabolismo , Aminocumarinas/química , Antibacterianos/química , Ácidos Dicarboxílicos/química , Deleção de Genes , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirróis/química , Streptomyces/enzimologiaRESUMO
MbtH-like proteins consist of â¼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes.
Assuntos
Aminocumarinas/metabolismo , Proteínas de Bactérias/metabolismo , Nucleotidiltransferases/metabolismo , Streptomyces coelicolor/enzimologia , Tirosina/metabolismo , Vancomicina/biossíntese , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação de Sentido Incorreto , Nucleotidiltransferases/genética , Streptomyces coelicolor/genética , Tirosina/genéticaRESUMO
The biosynthesis of aminocoumarin antibiotics requires two acyladenylate-forming enzymes: one for the activation of L-tyrosine as a precursor of the aminocoumarin moiety and another for the linkage of an acyl moiety to the aminocoumarin moiety. Unexpectedly, the biosynthetic gene cluster of the aminocoumarin antibiotic rubradirin was found to contain three genes for putative acyladenylate-forming enzymes of aminocoumarin biosynthesis and conjugation. We expressed, purified, and investigated these three proteins. Orf4 (55 kDa) was shown to be an active aminocoumarin acyl ligase. RubF6 (56 kDa) was inactive, but could be converted into an active enzyme by site-directed mutagenesis. RubC1 (138 kDa) was shown to be a unique bifunctional enzyme, comprising an aminocoumarin acyl ligase, and tyrosine-adenylation and peptidyl-carrier domains. This natural hybrid enzyme is unique among known proteins. A hypothesis is proposed as to how such an enzyme could offer a particularly effective machinery for aminocoumarin antibiotic biosynthesis.
Assuntos
Aminocumarinas/metabolismo , Ligases/química , Ligases/metabolismo , Streptomyces/enzimologia , Tirosina/metabolismo , Sequência de Aminoácidos , Biocatálise , Ativação Enzimática , Ligases/genética , Dados de Sequência Molecular , Naftoquinonas/metabolismo , Fases de Leitura AbertaRESUMO
Coumermycin A(1) is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It contains three pyrrole rings, that is, two terminal 5-methyl-pyrrole-2-carboxyl moieties and a central 3-methylpyrrole-2,4-dicarboxylic acid moiety. The biosynthesis of the terminal pyrrole moieties has been elucidated previously. However, the biosynthetic precursors of the central pyrrole moiety have remained unknown, and none of the genes or enzymes involved in its formation has been identified. We now show that five genes, contained in a contiguous 4.7 kb region within the coumermycin biosynthetic gene cluster, are required for the biosynthesis of this central pyrrole moiety. Each of these genes was deleted individually, resulting in a strong reduction or an abolishment of coumermycin production. External feeding of the central pyrrole moiety restored coumermycin production. One of these genes shows similarity to L-threonine kinase genes. Feeding of [U-(13)C,(15) N]L-threonine and (13)C NMR analysis of the resulting compound unequivocally proved that threonine was incorporated intact into the central pyrrole (19 % enrichment) to provide the heterocyclic nitrogen as well as four of the seven carbons of this moiety. Therefore, this pyrrole is formed via a new, hitherto unknown biosynthetic pathway. A hypothesis for the reaction sequence leading to the central pyrrole moiety of coumermycin A(1) is presented.
Assuntos
Aminocumarinas/metabolismo , Antibacterianos/biossíntese , Pirróis/metabolismo , Treonina/metabolismo , Aminocumarinas/química , Antibacterianos/química , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética , Família Multigênica , Pirróis/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The biosynthesis of aminocoumarin antibiotics involves the action of amide synthetases which construct amide bonds between aminocoumarins and various acyl moieties. Libraries of aminocoumarin analogues have been generated by in vivo fermentation, via feeding known amide synthetase substrates into producing microbial strains. Critically, such feeding studies rely on the inherent or engineered substrate promiscuity of each amide synthetase. We have initiated a program of directed evolution in order to create mutant amide synthetases for the synthesis of new nonnatural amino coumarin analogues. We used the clorobiocin enzyme CloL as a model amide synthetase to design and validate a fluorimetric high-throughput screen, which can be used to report the activity of mutant amide synthetases toward a broad range of coumarin and acyl donor substrates. Our assay monitors the decrease in fluorescence of aminocoumarins on acylation. The utility of the assay was illustrated by screening a library of amide synthetase mutants created by error-prone PCR. The substrate specificity of an amide synthetase was also rapidly probed using this assay, affording several newly identified substrates. It is anticipated that this high-throughput screen will accelerate the creation of amide synthetase mutants with new specificities by directed evolution.
Assuntos
Amida Sintases/metabolismo , Aminocumarinas/metabolismo , Evolução Molecular Direcionada/métodos , Ensaios de Triagem em Larga Escala/métodos , Novobiocina/análogos & derivados , Acilação , Aminocumarinas/química , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescência , Fluorometria/métodos , Estrutura Molecular , Novobiocina/química , Novobiocina/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
BACKGROUND: Urease enzyme catalyzes the hydrolysis of urea into ammonia and CO2, excess ammonia causes global warming and crop reduction. Ureases are also responsible for certain human diseases such as stomach cancer, peptic ulceration, pyelonephritis, and kidney stones. New urease inhibitors are developed to get rid of such problems. OBJECTIVE: This article describes the synthesis of a series of novel 1-aroyl-3-(2-oxo-2H-chromen-4- yl)thiourea derivatives (5a-j) as Jack bean urease inhibitors. METHODS: Freshly prepared aryl isothiocyanates were reacted with 4-aminocoumarin in the same pot in an anhydrous medium of acetone. The structures of the title thioureas (5a-j) were ascertained by their spectroscopic data. The inhibitory effects against jack bean urease were determined. RESULTS: It was found that compounds 5i and 5j showed excellent activity with IC50 values 0.0065 and 0.0293, µM respectively. Compound 5i bearing 4-methyl substituted phenyl ring plays a vital role in enzyme inhibitory activity. The kinetic mechanism analyzed by Lineweavere-Burk plots revealed that compound 5i inhibits the enzyme non-competitively. The Michaelis-Menten constant Km and inhibition constants Ki calculated from Lineweavere-Burk plots for compound 5i are 4.155mM and 0.00032µM, respectively. The antioxidant activity results displayed that compound 5j showed excellent radical scavenging activity. The cytotoxic effects determined against brine shrimp assay showed that all of the synthesized compounds are non-toxic to shrimp larvae. Molecular docking studies were performed against target protein (PDBID 4H9M) and it was determined that most of the synthesized compounds exhibited good binding affinity with the target protein. Molecular dynamics simulation (MDS) results revealed that compound 5i forms a stable complex with target protein showing little fluctuation. CONCLUSIONS: Based upon our investigations, it is proposed that 5i derivative may serve as a lead structure for devising more potent urease inhibitors.
Assuntos
Aminocumarinas/síntese química , Aminocumarinas/farmacologia , Canavalia/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Urease/antagonistas & inibidores , Aminocumarinas/química , Aminocumarinas/metabolismo , Animais , Artemia , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Conformação Proteica , Urease/química , Urease/metabolismoRESUMO
Coumermycin A1 is a natural aminocoumarin that inhibits bacterial DNA gyrase, a member of the GHKL proteins superfamily. We report here the first cocrystal structures of gyrase B bound to coumermycin A1, revealing that one coumermycin A1 molecule traps simultaneously two ATP-binding sites. The inhibited dimers from different species adopt distinct sequence-dependent conformations, alternative to the ATP-bound form. These structures provide a basis for the rational development of coumermycin A1 derivatives for antibiotherapy and biotechnology applications.
Assuntos
Aminocumarinas/química , DNA Girase/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Aminocumarinas/metabolismo , Sítios de Ligação , DNA Girase/metabolismo , Dimerização , Escherichia coli/enzimologia , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Thermus thermophilus/enzimologiaRESUMO
Many secondary metabolites of clinical importance have been isolated from different Streptomyces species. As most of the natural producers remain difficult to handle genetically, heterologous expression of an entire biosynthetic gene cluster in a well characterised host allows improved possibilities for modifications of the desired compound by manipulation of the biosynthetic genes. However, the large size of a functional gene cluster often prevents its direct cloning into a single cosmid clone. Here we describe a successful strategy to assemble the entire coumermycin A1 biosynthetic gene cluster (38.6 kb) into a single cosmid clone by lambda RED recombination technology. Heterologous expression of the reconstituted gene cluster in Streptomyces coelicolor M512 resulted in the heterologous production of coumermycin A1. Inactivation of the methyltransferase gene couO--responsible for the C-methylation at the 8-positions of the aminocoumarin moieties in coumermycin A1--and heterologous expression of the modified cluster resulted in an accumulation of a C-8-unsubstituted coumermycin A1 derivative. Subsequent expression of the halogenase gene clo-hal from the clorobiocin gene cluster in the heterologous producer strain led to the formation of two new hybrid antibiotics, containing either one or two chlorine atoms. The identities of the new compounds were verified by LC-MS, and their antibacterial activities were tested against Bacillus subtilis in an agar diffusion assay.
Assuntos
Expressão Gênica , Família Multigênica/genética , Aminocumarinas/química , Aminocumarinas/metabolismo , Aminocumarinas/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Bases de Dados Genéticas , Regulação da Expressão Gênica , Engenharia Genética , Espectrometria de Massas , Metiltransferases/genética , Metiltransferases/metabolismo , Estrutura Molecular , Mutação/genética , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Three new aminocoumarin antibiotics, termed ferulobiocin, 3-chlorocoumarobiocin and 8'-dechloro-3-chlorocoumarobiocin, were isolated from the culture broth of a Streptomyces coelicolor M512 strain expressing a modified clorobiocin biosynthetic gene cluster. Structural analysis showed that these new aminocoumarins were very similar to clorobiocin, with a substituted 4-hydroxycinnamoyl moieties instead of the prenylated 4-hydroxybenzoyl moiety of clorobiocin. The possible biosynthetic origin of these moieties is discussed.
Assuntos
Aminocumarinas/metabolismo , Antibacterianos/biossíntese , Ácidos Cumáricos/metabolismo , Genes Bacterianos/genética , Família Multigênica/genética , Novobiocina/análogos & derivados , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Aminocumarinas/química , Aminocumarinas/isolamento & purificação , Antibacterianos/química , Antibacterianos/isolamento & purificação , Meios de Cultura/metabolismo , Expressão Gênica , Testes de Sensibilidade Microbiana , Novobiocina/biossíntese , Propionatos , Streptomyces coelicolor/crescimento & desenvolvimentoRESUMO
Heat shock protein (Hsp) 90 an emerging and attracting target in the anti-HIV drug discovery process due to the key role it plays in the pathogenicity of HIV-1 virus. In this research study, long-range all-atom molecular dynamics simulations were engaged for the bound and the unbound proteins to enhance the understanding of the molecular mechanisms of the Hsp90 dimerization and inhibition. Results evidently showed that coumermycin A1 (C-A1), a recently discovered Hsp90 inhibitor, binds at the dimer's active site of the Hsp90 protein and leads to a substantial parting between dimeric opposed residues, which include Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669.A. Significant differences in magnitudes were observed in radius of gyration, root-mean-square deviation and root-mean-square fluctuation, which confirms a reasonably more flexible state in the apo conformation associated with it dimerization. In contrast, the bound conformer of Hsp90 showed less flexibility. This visibly highpoints the inhibition process resulting from the binding of the ligand. These findings were further validated by principal component analysis. We believe that the detailed dynamic analyses of Hsp90 presented in this study, would give an imperative insight and better understanding to the function and mechanisms of inhibition. Furthermore, information obtained from the binding mode of the inhibitor would be of great assistance in the design of more potent inhibitors against the HIV target Hsp90.
Assuntos
Aminocumarinas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Aminocumarinas/química , Sítios de Ligação , Dimerização , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Simulação de Dinâmica Molecular , Análise de Componente Principal , Ligação Proteica , TermodinâmicaAssuntos
Aminocumarinas/metabolismo , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Inibidores da Topoisomerase II , Aminocumarinas/química , Antibacterianos/química , Estrutura Molecular , Novobiocina/análogos & derivados , Novobiocina/biossíntese , Novobiocina/química , Novobiocina/metabolismoRESUMO
Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a 'switch' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.
Assuntos
Apoptose/genética , Multimerização Proteica/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Recombinantes/metabolismo , Aminocumarinas/metabolismo , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.
Assuntos
Aminocumarinas/metabolismo , Bioensaio , Catepsina C/análise , Dipeptídeos/metabolismo , Corantes Fluorescentes/metabolismo , Lisossomos/enzimologia , Animais , Linhagem Celular Tumoral , Fluorescência , Humanos , Espaço Intracelular/enzimologia , Especificidade por SubstratoRESUMO
BACKGROUND: Neurotrophins elicit both acute and long-term modulation of synaptic transmission and plasticity. Previously, we demonstrated that the long-term synaptic modulation requires the endocytosis of neurotrophin-receptor complex, the activation of PI3K and Akt, and mTOR mediated protein synthesis. However, it is unclear whether the long-term synaptic modulation by neurotrophins depends on protein synthesis in pre- or post-synaptic cells. RESULTS: Here we have developed an inducible protein translation blocker, in which the kinase domain of protein kinase R (PKR) is fused with bacterial gyrase B domain (GyrB-PKR), which could be dimerized upon treatment with a cell permeable drug, coumermycin. By genetically targeting GyrB-PKR to specific cell types, we show that NT-3 induced long-term synaptic modulation requires presynaptic, but not postsynaptic protein synthesis. CONCLUSIONS: Our results provide mechanistic insights into the cell-specific requirement for protein synthesis in the long-term synaptic modulation by neurotrophins. The GyrB-PKR system may be useful tool to study protein synthesis in a cell-specific manner.
Assuntos
Plasticidade Neuronal/fisiologia , Neurotrofina 3/metabolismo , Terminações Pré-Sinápticas/fisiologia , Biossíntese de Proteínas , Transmissão Sináptica/fisiologia , eIF-2 Quinase/química , Aminocumarinas/metabolismo , Animais , DNA Girase/química , DNA Girase/genética , DNA Girase/metabolismo , Dimerização , Potenciação de Longa Duração/fisiologia , Junção Neuromuscular/anatomia & histologia , Junção Neuromuscular/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neurotrofina 3/genética , Técnicas de Patch-Clamp , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas de Cultura de Tecidos , Inibidores da Topoisomerase II , Xenopus laevis/embriologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
A new strategy for affinity-based drug delivery by modification of the drug rather than modification of the device is presented. Rifampin is modified to contain either one or two PEG-adamantane arms, and the drug release properties of dimeric coumermycin are compared to novobiocin with only one biding domain. The drugs are loaded into affinity-based and diffusion-only delivery platforms, the loading efficiency is calculated, and the release kinetics is determined in vitro. The presence of additional binding domains prolongs the release of antibiotics. Release rates differ little between modified and unmodified drug from the diffusion-only system. The results demonstrate the feasibility of custom-tuning drug delivery by multiplexing interactions with an affinity-based polymer platform.
Assuntos
Ciclodextrinas/química , Preparações de Ação Retardada/síntese química , Portadores de Fármacos/síntese química , Composição de Medicamentos/métodos , Rifampina/química , Adamantano/química , Aminocumarinas/química , Aminocumarinas/metabolismo , Aminocumarinas/farmacologia , Sítios de Ligação , Preparações de Ação Retardada/metabolismo , Difusão , Portadores de Fármacos/metabolismo , Humanos , Hidrogéis/química , Cinética , Novobiocina/química , Novobiocina/metabolismo , Novobiocina/farmacologia , Polietilenoglicóis/química , Rifampina/metabolismo , Rifampina/farmacologia , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimentoRESUMO
The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A(1) and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.8-fold increase of productivity. Presumably, this copolymer acts as an oxygen carrier. The additional inclusion of cobalt chloride (0.2-2 mg l(-1)) dramatically increased the percentage of the desired compound clorobiocin within the total produced clorobiocin derivatives. This is very likely due to a stimulation of a cobalamin-dependent methylation reaction catalyzed by the enzyme CloN6 of clorobiocin biosynthesis. All three investigated host strains (S. coelicolor M512, M1146 and M1154) gave similar production rates of total clorobiocin derivatives (on average, 158 mg l(-1) in the presence of 0.6% Q2-5247 and 0.2 mg l(-1) CoCl(2)). In contrast, heterologous production of caprazamycin derivatives was optimal in strain M1154 (amounts of 152 mg l(-1) on average).