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1.
Nat Commun ; 12(1): 545, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483499

RESUMO

In Rhizobiales bacteria, such as Sinorhizobium meliloti, cell elongation takes place only at new cell poles, generated by cell division. Here, we show that the role of the FtsN-like protein RgsS in S. meliloti extends beyond cell division. RgsS contains a conserved SPOR domain known to bind amidase-processed peptidoglycan. This part of RgsS and peptidoglycan amidase AmiC are crucial for reliable selection of the new cell pole as cell elongation zone. Absence of these components increases mobility of RgsS molecules, as well as abnormal RgsS accumulation and positioning of the growth zone at the old cell pole in about one third of the cells. These cells with inverted growth polarity are able to complete the cell cycle but show partially impaired chromosome segregation. We propose that amidase-processed peptidoglycan provides a landmark for RgsS to generate cell polarity in unipolarly growing Rhizobiales.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Sinorhizobium meliloti/metabolismo , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Western Blotting , Divisão Celular/genética , Proteínas de Membrana/genética , Microscopia de Fluorescência , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crescimento & desenvolvimento
2.
Mol Immunol ; 127: 47-55, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32927163

RESUMO

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can infect and replicate in macrophages. Peptidoglycan (PGN) is a major component of the mycobacterial cell wall and is recognized by host pattern recognition receptors (PRRs). Many bacteria modulate and evade the immune defenses of their hosts through PGN deacetylation. Rv1096 was previously characterized as a PGN N-deacetylase gene in Mtb. However, the underlying mechanism by which Rv1096 regulates host immune defenses during macrophage infection remains unclear. In the present study, we investigated the role of Rv1096 in evading host immunity using a recombinant M. smegmatis expressing exogenous Rv1096 and Rv1096-deleted Mtb strain H37Rv mutant. We found that Rv1096 promoted intracellular bacillary survival and inhibited the inflammatory response in M. smegmatis- or Mtb-infected macrophages. The inhibition of mycobacteria-induced inflammatory response in macrophages was at least partially due to NF-κB and MAPK activation downstream of TLR and NOD signaling pathways. Furthermore, we found that Rv1096 inhibitory effect on inflammatory response was associated with TLR2, TLR4 and NOD2. Finally, we demonstrated the PGN deacetylase activity of Rv1096 by Fourier transform IR and Rv1096 NODB deficient mutant. Our findings suggest that Rv1096 may deacetylate PGNs to evade PRRs recognition, thus protecting Mtb from host immune surveillance and clearance in macrophages.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Sistema de Sinalização das MAP Quinases , Viabilidade Microbiana , Mycobacterium smegmatis/citologia , Mycobacterium tuberculosis/enzimologia , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Animais , Proteínas de Bactérias/química , Citocinas/metabolismo , Feminino , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Domínios Proteicos , Células RAW 264.7
3.
PLoS One ; 15(7): e0235643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735615

RESUMO

BACKGROUND: Pyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function. OBJECTIVE: To develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene. METHODS: We measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models. RESULTS: The stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation. CONCLUSIONS: This study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Amidoidrolases/química , Amidoidrolases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Cinética , Modelos Lineares , Simulação de Dinâmica Molecular , Mutagênese , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
4.
Am J Physiol Heart Circ Physiol ; 319(3): H582-H603, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32762558

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.


Assuntos
Arginina/análogos & derivados , Cardiomiopatias/metabolismo , Circulação Coronária , Tolerância ao Exercício , Heterozigoto , Distrofia Muscular de Duchenne/metabolismo , Miocárdio/metabolismo , Músculo Quadríceps/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Arginina/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/patologia , Necrose , Músculo Quadríceps/patologia , Músculo Quadríceps/fisiopatologia , Função Ventricular Esquerda
5.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
6.
J Med Chem ; 63(13): 6959-6978, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32551649

RESUMO

Human mitochondrial peptide deformylase (HsPDF) is responsible for removing the formyl group from N-terminal formylmethionines of newly synthesized mitochondrial proteins and plays important roles in maintaining mitochondria function. It is overexpressed in various cancers and has been proposed as a novel therapeutic target. Actinonin, a naturally occurring peptidomimetic HsPDF inhibitor, was reported to inhibit the proliferation of a broad spectrum of human cancer cells in vitro. However, its efficacy and pharmacokinetic profile requires significant improvement for therapeutic purposes. To obtain HsPDF inhibitors as anticancer therapeutics, we screened an in-house collection of actinonin derivatives and found two initial hits with antiproliferation activity. Further optimization along the peptidomimetic backbone lead to two series of compounds containing substituted phenyl moieties. They are potent HsPDF inhibitors and exhibited greatly improved antiproliferation activity in selected cancer cell lines. Finally, compound 15m significantly inhibited the growth of human colon cancer in xenograft animal models.


Assuntos
Amidoidrolases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Amidoidrolases/química , Amidoidrolases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HCT116 , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Exp Anim ; 69(4): 388-394, 2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-32507787

RESUMO

We recently demonstrated that aspartoacylase (Aspa) and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) genes were causative of essential tremor (ET) in rats. This finding was obtained using Aspaem34Kyo/Hcn1A354V double-mutant rats, but they were bred on a heterogeneous genetic background of two strains, F344 and WTC. Here, we developed an Aspaem34Kyo/Hcn1em1Kyo double-knockout rat strain with a homogenous F344 genetic background and studied the ability of glutamate receptor antagonists to suppress ET. The F344-Aspa/Hcn1 double-knockout rats exhibited spontaneous, intense body tremor equivalent to that in the double-mutant rats. N-acetyl-aspartate (NAA), a substrate of ASPA, showed accumulation in all brain regions and in the spinal cord. However, N-acetyl-aspartyl-glutamate (NAAG), which is derived from NAA and interacts with glutamatergic receptors, was decreased in the medulla oblongata of the double-knockout rats. The tremor was suppressed by 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, in F344-Aspa/Hcn1 double-knockout rats. The non-NMDA glutamate receptor antagonist NBQX weakly inhibited the tremor, while the metabotropic glutamate receptor antagonist LY341495 showed no effect. In addition, both NR2B subunit-specific (Ro 25-6981) and NR2C/NR2D subunit-specific (cis-piperidine dicarboxylic acid) NMDA receptor antagonists suppressed the tremor. These data indicated that the pathogenesis of tremor in Aspa/Hcn1 double-knockout rats involved ionotropic glutamate receptors, particularly NMDA receptors.


Assuntos
Amidoidrolases/genética , Tremor Essencial/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais de Potássio/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/fisiologia , Amidoidrolases/metabolismo , Animais , Encéfalo/metabolismo , Tremor Essencial/tratamento farmacológico , Técnicas de Inativação de Genes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Terapia de Alvo Molecular , Fenóis/farmacologia , Fenóis/uso terapêutico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Canais de Potássio/metabolismo , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Ratos Endogâmicos F344 , Ratos Mutantes , Medula Espinal/metabolismo
8.
Nat Commun ; 11(1): 2450, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415073

RESUMO

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Polissacarídeos/metabolismo , Acetilação , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Domínio Catalítico , Sequência Conservada , Regulação Fúngica da Expressão Gênica , Glicosaminoglicanos/biossíntese , Metais/metabolismo , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Tempo
9.
Enzyme Microb Technol ; 137: 109536, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423673

RESUMO

N-acylated amino acids are widely used as surfactants and/or actives in cosmetics and household formulations. Their industrial production is based on the use of the Schotten-Baumann chemical and unselective reaction. Faced to the growing demand for greener production processes, selective enzymatic synthesis in more environment-friendly conditions starts to be considered as a potential alternative. This study concerns the use of the aminoacylases from Streptomyces ambofaciens to selectively catalyse aminoacid acylation reaction by fatty acids in aqueous medium. The results demonstrated that, when using undecylenoic acid as acyl donor, these aminoacylases properly catalyse the acylation of 14 of the 20 proteogenic l-amino acids tested on their α amino group with a great variability depending on the nature of the amino acid (polar or not, positively/negatively charged, aromatic or not…). More precisely, the following 9 amino acids were shown to be preferentially acylated by S. ambofaciens aminoacylases as follows: lysine > arginine > leucine > methionine > phenylalanine > valine > cysteine > isoleucine > threonine. Different fatty acids were used as acyl donors and, in most cases, the fatty acid length influenced the conversion yield. The kinetic study of α-lauroy-lysine synthesis showed a positive influence of lysine concentration with Vmax and Km of 3.7 mM/h and 76 mM, respectively. Besides, the lauric acid had an inhibitory effect on the reaction with Ki of 70 mM. The addition of cobalt to the reaction medium led to a more than six-fold increase of the reaction rate. These results, achieved with the aminoacylases from S. ambofaciens represent an improved enzyme-based N-acylated amino acids production in order to provide an alternative way to the Schotten-Baumann chemical reaction currently used in the industry.


Assuntos
Amidoidrolases/metabolismo , Aminoácidos/metabolismo , Biocatálise , Streptomyces/enzimologia , Acilação , Cobalto/metabolismo , Cinética
10.
Proc Natl Acad Sci U S A ; 117(20): 10778-10788, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366662

RESUMO

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate's degradation. We discovered that the human Nt-Asn-specific Nt-amidase NTAN1, Nt-Gln-specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate's dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron-targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed "superchanneling." In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate's sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex.


Assuntos
Proteólise , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação , Amidoidrolases/metabolismo , Aminoaciltransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
11.
Nat Chem Biol ; 16(6): 667-675, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393901

RESUMO

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.


Assuntos
Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Medo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Receptores de Canabinoides/metabolismo , Transdução de Sinais
12.
Nat Commun ; 11(1): 1938, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321911

RESUMO

Bacteria can produce membranous nanotubes that mediate contact-dependent exchange of molecules among bacterial cells. However, it is unclear how nanotubes cross the cell wall to emerge from the donor or to penetrate into the recipient cell. Here, we report that Bacillus subtilis utilizes cell wall remodeling enzymes, the LytC amidase and its enhancer LytB, for efficient nanotube extrusion and penetration. Nanotube production is reduced in a lytBC mutant, and the few nanotubes formed appear deficient in penetrating into target cells. Donor-derived LytB molecules localize along nanotubes and on the surface of nanotube-connected neighbouring cells, primarily at sites of nanotube penetration. Furthermore, LytB from donor B. subtilis can activate LytC of recipient bacteria from diverse species, facilitating cell wall hydrolysis to establish nanotube connection. Our data provide a mechanistic view of how intercellular connecting devices can be formed among neighbouring bacteria.


Assuntos
Amidoidrolases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Extensões da Superfície Celular/metabolismo , Parede Celular/enzimologia , Conjugação Genética , Amidoidrolases/genética , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Extensões da Superfície Celular/genética , Parede Celular/química , Parede Celular/genética , Parede Celular/metabolismo , Transporte Proteico
13.
J Pharmacol Exp Ther ; 373(3): 353-360, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32241809

RESUMO

Fatty acid amide hydrolase (FAAH) is a key enzyme in the endocannabinoid system. N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide ([11C]DFMC) was developed as an irreversible-type positron emission tomography (PET) tracer for FAAH. Here, we attempted to noninvasively estimate rate constant k3 (rate of transfer to the specifically-bound compartment) as a direct index for FAAH in the rat brain. First, the two-tissue compartment model analysis including three parameters [K1-k3, two-tissue compartment model for the irreversible-type radiotracer (2TCMi)] in PET study with [11C]DFMC was conducted, which provided 0.21 ± 0.04 ml·cm-3·min-1 of the net uptake value (Ki), an indirect index for FAAH, in the FAAH-richest region (the cingulate cortex). Subsequently, to noninvasively estimate Ki value, the reference model analysis (Patlak graphical analysis reference model) was tried using a time-activity curve of the spinal cord. In that result, the noninvasive Ki value (KREF) was concisely estimated with high correlation (r > 0.95) to Ki values based on 2TCMi. Using estimated KREF value, we tried to obtain calculated-k3 based on previously defined equations. The calculated k3 was successfully estimated with high correlation (r = 0.95) to direct k3 in 2TCMi. Finally, the dose relationship study using calculated k3 demonstrated that in vivo ED50 value of [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate, a major inhibitor of FAAH, was 66.4 µg/kg in rat brain. In conclusion, we proposed the calculated k3 as an alternative index corresponding to regional FAAH concentrations and suggested that PET with [11C]DFMC enables occupancy study for new pharmaceuticals targeting FAAH. SIGNIFICANCE STATEMENT: In the present study, we proposed calculated k3 as an alternative index corresponding with fatty acid amide hydrolase concentration. By using calculated k3, in vivo ED50 of [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate was successfully estimated to be 66.4 µg/kg for rats. Thus, we demonstrated the pharmacological utility of positron emission tomography with N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide.


Assuntos
Amidoidrolases/metabolismo , Encéfalo/metabolismo , Carbamatos/farmacologia , Radioisótopos de Carbono/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Roedores/metabolismo , Animais , Endocanabinoides/metabolismo , Masculino , Compostos Radiofarmacêuticos/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/fisiologia
14.
Int J Mol Sci ; 21(7)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244803

RESUMO

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval-pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval-pupal and pupal-adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.


Assuntos
Amidoidrolases/genética , Besouros/genética , Proteínas de Insetos/genética , Metamorfose Biológica/genética , Muda/genética , Amidoidrolases/classificação , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Quitina/metabolismo , Besouros/enzimologia , Besouros/crescimento & desenvolvimento , Ecdisterona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Insetos/metabolismo , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Filogenia , Pupa/enzimologia , Pupa/genética , Pupa/crescimento & desenvolvimento , Interferência de RNA , Asas de Animais/anormalidades , Asas de Animais/metabolismo
15.
J Biosci Bioeng ; 130(2): 115-120, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32253090

RESUMO

Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%-40% ethanol solutions. The optimum temperature of CPUTNase was 43°C. This enzyme showed urethanase activity at pH 5.5-10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase.


Assuntos
Amidoidrolases/isolamento & purificação , Amidoidrolases/metabolismo , Candida parapsilosis/enzimologia , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Candida parapsilosis/genética , Cromatografia por Troca Iônica , Estabilidade Enzimática/efeitos dos fármacos , Etanol/farmacologia , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Uretana/metabolismo
16.
J Biosci Bioeng ; 130(1): 82-88, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32280054

RESUMO

Acylase is known as a quorum quenching enzyme that degrades N-acyl-homoserine lactones (AHLs), a key signaling molecule in a quorum sensing (QS) mechanism. Acylase I cleaves the acyl-chain in the chemical structures of AHLs, thereby exerting an anti-biofilm effect by the inhibition of bacterial cell-cell communication and resultant secretion of extracellular polymeric substances (EPS). However, the physical and physiological impacts of acylase on bacterial cells remain to be systematically elucidated. This study, therefore, investigated the effect of active and inactive acylase addition on the growth, viability, and cell morphologies of Agrobacterium tumefaciens. For comparison, active and inactive lysozymes were taken as positive controls. The results showed that active acylase inhibited A. tumefaciens cell growth at concentrations ranging from 0.1 to 1000 µg mL-1, and so did active lysozyme. Fluorescent detection by Live/Dead staining underpinned that cell viability of A. tumefaciens decreased at concentrations higher than 0.1 µg mL-1 for both acylase and lysozyme, although lysozyme inflicted higher degree of cellular damage. Moreover, atomic force microscopy unraveled a noticeable distortion of A. tumefaciens cells by both acylase and lysozyme. Together, the results showed that acylase not only blocked AHLs-based QS mechanisms but also compromised cell viability and altered surface morphology of A. tumefaciens cells, as observed by the addition of hydrolase.


Assuntos
Agrobacterium tumefaciens/enzimologia , Agrobacterium tumefaciens/crescimento & desenvolvimento , Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Acil-Butirolactonas/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/fisiologia , Amidoidrolases/genética , Proteínas de Bactérias/genética , Biofilmes , Percepção de Quorum
17.
Arch Insect Biochem Physiol ; 104(1): e21666, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32112466

RESUMO

Chitin deacetylase (CDA) is a hydrolytic enzyme that modifies chitin into chitosan in the body of insects. In this study, we obtained a full-length complementary DNA sequence (MsCDA1) from the oriental armyworm Mythimna separata by high-throughput sequencing. MsCDA1 is 1,952 bp long and includes 1,620 bp open reading frame encoding 539 amino acids. Analysis by quantitative real time polymerase chain reaction showed that MsCDA1 expression was higher at the adult stage than at earlier developmental stages. MsCDA1 was expressed in all larval tissues examined, in which the highest expression level was found in the midgut. The RNA interference (RNAi) suppressed MsCDA1 expression levels at 12, 24, and 48 hr after injection of double-stranded RNA (1-4 µg per larva) specific to MsCDA1. Under RNAi condition, CDA enzyme activity was significantly reduced and changes an ultramicroscopic structure of M. separata peritrophic matrix especially in its microfibrillar organization exhibiting loose network. In contrast, the surface of the peritrophic matrix was relatively smooth and well organized at control or low RNAi conditions. Moreover, RNAi of MsCDA1 expression impaired larval growth and development, occasionally leading to larval death. These results demonstrate that MsCDA1 plays a crucial role in maintaining peritrophic matrix integrity in M. separata.


Assuntos
Amidoidrolases/genética , Mariposas/enzimologia , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Quitina/metabolismo , Trato Gastrointestinal/ultraestrutura , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Interferência de RNA , Análise de Sequência de DNA
18.
Oxid Med Cell Longev ; 2020: 1015908, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215167

RESUMO

Despite the development of new drugs and other therapeutic strategies, cardiovascular disease (CVD) remains still the major cause of morbidity and mortality in the world population. A lot of research, performed mostly in the last three decades, revealed an important correlation between "classical" demographic and biochemical risk factors for CVD, (i.e., hypercholesterolemia, hyperhomocysteinemia, smoking, renal failure, aging, diabetes, and hypertension) with endothelial dysfunction associated directly with the nitric oxide deficiency. The discovery of nitric oxide and its recognition as an endothelial-derived relaxing factor was a breakthrough in understanding the pathophysiology and development of cardiovascular system disorders. The nitric oxide synthesis pathway and its regulation and association with cardiovascular risk factors were a common subject for research during the last decades. As nitric oxide synthase, especially its endothelial isoform, which plays a crucial role in the regulation of NO bioavailability, inhibiting its function results in the increase in the cardiovascular risk pattern. Among agents altering the production of nitric oxide, asymmetric dimethylarginine-the competitive inhibitor of NOS-appears to be the most important. In this review paper, we summarize the role of L-arginine-nitric oxide pathway in cardiovascular disorders with the focus on intraplatelet metabolism.


Assuntos
Arginina/análogos & derivados , Plaquetas/metabolismo , Doenças Cardiovasculares/sangue , Redes e Vias Metabólicas , Óxido Nítrico/metabolismo , Amidoidrolases/metabolismo , Arginina/sangue , Arginina/metabolismo , Doenças Cardiovasculares/metabolismo , Endotélio Vascular/metabolismo , Humanos , Óxido Nítrico/sangue , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Fatores de Risco
19.
J Enzyme Inhib Med Chem ; 35(1): 815-823, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32200655

RESUMO

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here, we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibitors. N-(3-Bromopyridin-2-yl)-2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the hydrolysis of [3H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhibition with a Ki value of 0.26 µM and an α value of 4.9. At a concentration of 10 µM, the compound did not inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2. However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cyclooxygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting FAAH/substrate-selective COX inhibitor.


Assuntos
Amidas/farmacologia , Amidoidrolases/antagonistas & inibidores , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Ibuprofeno/farmacologia , Amidas/síntese química , Amidas/química , Amidoidrolases/metabolismo , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Ibuprofeno/síntese química , Ibuprofeno/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Relação Estrutura-Atividade
20.
Sci Rep ; 10(1): 4329, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152368

RESUMO

Chitin deacetylase (CDA) can hydrolyse the acetamido group of chitin polymers to produce chitosans, which are used in various fields including the biomedical and pharmaceutical industries, food production, agriculture, and water treatment. CDA represents a more environmentally-friendly and easier to control alternative to the chemical methods currently utilised to produce chitosans from chitin; however, the majority of identified CDAs display activity toward low-molecular-weight oligomers and are essentially inactive toward polymeric chitin or chitosans. Therefore, it is important to identify novel CDAs with activity toward polymeric chitin and chitosans. In this study, we isolated the bacterium Rhodococcus equi F6 from a soil sample and showed that it expresses a novel CDA (ReCDA), whose activity toward 4-nitroacetanilide reached 19.20 U/mL/h during fermentation and was able to deacetylate polymeric chitin, colloidal chitin, glycol-chitin, and chitosan. Whole genome sequencing revealed that ReCDA is unique to the R. equi F6 genome, while phylogenetic analysis indicated that ReCDA is evolutionarily distant from other CDAs. In conclusion, ReCDA isolated from the R. equi F6 strain expands the known repertoire of CDAs and could be used to deacetylate polymeric chitosans and chitin in industrial applications.


Assuntos
Genoma Bacteriano , Genômica , Rhodococcus equi/classificação , Rhodococcus equi/genética , Acetilação , Amidoidrolases/biossíntese , Amidoidrolases/genética , Amidoidrolases/metabolismo , Biopolímeros , Quitosana/metabolismo , Genômica/métodos , Fenótipo , Filogenia , Rhodococcus equi/enzimologia , Rhodococcus equi/isolamento & purificação , Sequenciamento Completo do Genoma
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