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1.
Methods Mol Biol ; 2576: 241-247, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152192

RESUMO

Fatty acid amide hydrolase (FAAH) is an intracellular enzyme responsible for the hydrolysis of endogenous anandamide (AEA), a reaction that terminates the biological effects of this lipid mediator. The final products of AEA cleavage are arachidonic acid and ethanolamine. In the method described herein, FAAH activity is measured through the use of the radioactive substrate [14C-ethanolamine]-AEA and subsequent quantification of the reaction product [14C]-ethanolamine.


Assuntos
Amidoidrolases , Endocanabinoides , Amidoidrolases/metabolismo , Ácido Araquidônico , Encéfalo/metabolismo , Etanolamina , Etanolaminas , Hidrólise
2.
Methods Mol Biol ; 2576: 249-260, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152193

RESUMO

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the degradation of anandamide (N-arachidonoylethanolamine, AEA) to arachidonic acid (AA) and ethanolamine. The method described here measures FAAH activity through the fluorometric arachidonoyl-7-amino-4-methyl-coumarin amide (AAMCA) substrate, which allows a simple and sensitive assay suitable for high-throughput screening tests. FAAH catalyzes the hydrolysis of AAMCA producing AA and the highly fluorescent compound 7-amino-4-methylcoumarin (AMC).


Assuntos
Amidoidrolases , Alcamidas Poli-Insaturadas , Amidoidrolases/metabolismo , Ácido Araquidônico , Ácidos Araquidônicos , Cumarínicos , Endocanabinoides , Etanolaminas , Alcamidas Poli-Insaturadas/metabolismo
3.
J Steroid Biochem Mol Biol ; 225: 106180, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36243205

RESUMO

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) mainly degrades asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor. Emerging evidence suggested that plasma ADMA is accumulated in patients with polycystic ovary syndrome (PCOS). However, ADMA-DDAH1 involvement in PCOS pathogenesis is unclear. Here, we used dehydroepiandrosterone (DHEA)-induced PCOS rats and the ovarian granulosa cell line KGN to investigate the effect of the ADMA-DDAH1 pathway on ovarian apoptosis. Moreover, we also quantified the ADMA levels and redox status in human serum specimens, Sprague Dawley rats and KGN cells to investigate the effect of ADMA-DDAH1 on redox status and ovarian apoptosis in PCOS. We enrolled 19 women with PCOS and 17 healthy women (controls) in this study. The women with PCOS had increased serum ADMA levels and decreased glutathione peroxidase (GSH-PX) compared with the controls. In Sprague Dawley rats, 21-day DHEA treatment established PCOS and the rat contained higher ADMA levels in serum and lower DDAH1 expression in ovaries. Moreover, the PCOS rat serum and ovaries exhibited increased levels of the oxidative stress marker malondialdehyde (MDA). ADMA treatment of the KGN cells induced reactive oxygen species accumulation and led to apoptosis. Contrastingly, overexpressing DDAH1 in the KGN cells significantly decreased ADMA levels, enhanced cell viability, and inhibited oxidative stress, while the effect was inverse in DDAH1 knockdown cells. Overall, our results demonstrated that PCOS involves elevated ADMA levels and redox imbalance. The ADMA-DDAH1 pathway exerted a marked effect on oxidative stress and ovarian apoptosis in PCOS. Our findings suggested that strategies for increasing DDAH1 activity in ovarian cells may provide a novel approach for ameliorating PCOS.


Assuntos
Síndrome do Ovário Policístico , Humanos , Feminino , Ratos , Animais , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/metabolismo , Ratos Sprague-Dawley , Amidoidrolases/genética , Amidoidrolases/metabolismo , Transdução de Sinais , Arginina/metabolismo , Apoptose , Desidroepiandrosterona/farmacologia
4.
Gut Microbes ; 14(1): 2149023, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36420990

RESUMO

The mechanisms by which early microbial colonizers of the neonate influence gut development are poorly understood. Bacterial bile salt hydrolase (BSH) acts as a putative colonization factor that influences bile acid signatures and microbe-host signaling pathways and we considered whether this activity can influence infant gut development. In silico analysis of the human neonatal gut metagenome confirmed that BSH enzyme sequences are present as early as one day postpartum. Gastrointestinal delivery of cloned BSH to immature gnotobiotic mice accelerated shortening of the colon and regularized gene expression profiles, with monocolonised mice more closely resembling conventionally raised animals. In situ expression of BSH decreased markers of cell proliferation (Ki67, Hes2 and Ascl2) and strongly increased expression of ALPI, a marker of cell differentiation and barrier function. These data suggest an evolutionary paradigm whereby microbial BSH activity potentially influences bacterial colonization and in-turn benefits host gastrointestinal maturation.


Assuntos
Microbioma Gastrointestinal , Transcriptoma , Feminino , Humanos , Camundongos , Animais , Amidoidrolases/genética , Amidoidrolases/metabolismo , Trato Gastrointestinal/microbiologia , Bactérias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo
5.
Int J Mol Sci ; 23(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36232364

RESUMO

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the potential function of PGRPs in the giant triton snail Charonia tritonis. In this study, a short-type PGRP gene (termed Ct-PGRP-S1) was identified in C. tritonis. Ct-PGRP-S1 was predicted to contain several structural features known in PGRPs, including a typical PGRP domain (Amidase_2) and Src homology-3 (SH3) domain. The Ct-PGRP-S1 gene was constitutively expressed in all tissues examined except in proboscis, with the highest expression level observed in the liver. As a typical PRR, Ct-PGRP-S1 has an ability to degrade peptidoglycan (PGN) and was proven to have non-Zn2+-dependent amidase activity and antibacterial activity against Vibrioalginolyticus and Staphylococcus aureus. It is the first report to reveal the peptidoglycan recognition protein in C. tritonis, and these results suggest that peptidoglycan recognition protein Ct-PGRP-S1 is an important effector of C. tritonis that modulates bacterial infection resistance of V. alginolyticus and S. aureus, and this study may provide crucial basic data for the understanding of an innate immunity system of C. tritonis.


Assuntos
Peptidoglicano , Infecções Estafilocócicas , Amidoidrolases/metabolismo , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte , Clonagem Molecular , Imunidade Inata , Peptidoglicano/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Caramujos/genética , Staphylococcus aureus/metabolismo
6.
Int J Mol Sci ; 23(19)2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36233204

RESUMO

The contribution of nitric oxide synthases (NOSs) to the pathophysiology of several neuropsychiatric disorders is recognized, but the role of their regulators, dimethylarginine dimethylaminohydrolases (DDAHs), is less understood. This study's objective was to estimate DDAH1 and DDAH2 associations with biological processes implicated in major psychiatric disorders using publicly accessible expression databases. Since co-expressed genes are more likely to be involved in the same biologic processes, we investigated co-expression patterns with DDAH1 and DDAH2 in the dorsolateral prefrontal cortex in psychiatric patients and control subjects. There were no significant differences in DDAH1 and DDAH2 expression levels in schizophrenia or bipolar disorder patients compared to controls. Meanwhile, the data suggest that in patients, DDAH1 and DDHA2 undergo a functional shift mirrored in changes in co-expressed gene patterns. This disarrangement appears in the loss of expression level correlations between DDAH1 or DDAH2 and genes associated with psychiatric disorders and reduced functional similarity of DDAH1 or DDAH2 co-expressed genes in the patient groups. Our findings evidence the possible involvement of DDAH1 and DDAH2 in neuropsychiatric disorder development, but the underlying mechanisms need experimental validation.


Assuntos
Amidoidrolases , Produtos Biológicos , Transtornos Mentais , Amidoidrolases/genética , Amidoidrolases/metabolismo , Arginina/metabolismo , Humanos , Transtornos Mentais/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase
7.
Int J Mol Sci ; 23(20)2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36293543

RESUMO

Ischemic cardiomyopathy leads to inflammation and left ventricular (LV) dysfunction. Animal studies provided evidence for cardioprotective effects of the endocannabinoid system, including cardiomyocyte adaptation, inflammation, and remodeling. Cannabinoid type-2 receptor (CB2) deficiency led to increased apoptosis and infarctions with worsened LV function in ischemic cardiomyopathy. The aim of our study was to investigate a possible cardioprotective effect of endocannabinoid anandamide (AEA) after ischemia and reperfusion (I/R). Therefore, fatty acid amide hydrolase deficient (FAAH)-/- mice were subjected to repetitive, daily, 15 min, left anterior descending artery (LAD) occlusion over 3 and 7 consecutive days. Interestingly, FAAH-/- mice showed stigmata such as enhanced inflammation, cardiomyocyte loss, stronger remodeling, and persistent scar with deteriorated LV function compared to wild-type (WT) littermates. As endocannabinoids also activate PPAR-α (peroxisome proliferator-activated receptor), PPAR-α mediated effects of AEA were eliminated with PPAR-α antagonist GW6471 i.v. in FAAH-/- mice. LV function was assessed using M-mode echocardiography. Immunohistochemical analysis revealed apoptosis, macrophage accumulation, collagen deposition, and remodeling. Hypertrophy was determined by cardiomyocyte area and heart weight/tibia length. Molecular analyses involved Taqman® RT-qPCR and immune cells were analyzed with fluorescence-activated cell sorting (FACS). Most importantly, collagen deposition was reduced to WT levels when FAAH-/- mice were treated with GW6471. Chemokine ligand-2 (CCL2) expression was significantly higher in FAAH-/- mice compared to WT, followed by higher macrophage infiltration in infarcted areas, both being reversed by GW6471 treatment. Besides restoring antioxidative properties and contractile elements, PPAR-α antagonism also reversed hypertrophy and remodeling in FAAH-/- mice. Finally, FAAH-/--mice showed more substantial downregulation of PPAR-α compared to WT, suggesting a compensatory mechanism as endocannabinoids are also ligands for PPAR-α, and its activation causes lipotoxicity leading to cardiomyocyte apoptosis. Our study gives novel insights into the role of endocannabinoids acting via PPAR-α. We hypothesize that the increase in endocannabinoids may have partially detrimental effects on cardiomyocyte survival due to PPAR-α activation.


Assuntos
Canabinoides , Cardiomiopatias , Doença da Artéria Coronariana , Isquemia Miocárdica , Disfunção Ventricular Esquerda , Camundongos , Animais , Endocanabinoides/metabolismo , Ligantes , Amidoidrolases/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Alcamidas Poli-Insaturadas/metabolismo , Receptores de Canabinoides , PPAR alfa/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Inflamação , Reperfusão , Colágeno , Hipertrofia
8.
Biomolecules ; 12(10)2022 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-36291701

RESUMO

The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications.


Assuntos
Amidoidrolases , Pró-Fármacos , Humanos , Amidoidrolases/metabolismo , Nucleosídeos , Proteínas , Citosina , Pirimidinas/metabolismo , Especificidade por Substrato
9.
Bioorg Med Chem ; 74: 117046, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36228522

RESUMO

Tuberculosis (TB) remains a leading cause of infectious disease-related mortality and morbidity. Pyrazinamide (PZA) is a critical component of the first-line TB treatment regimen because of its sterilizing activity against non-replicating Mycobacterium tuberculosis (Mtb), but its mechanism of action has remained enigmatic. PZA is a prodrug converted by pyrazinamidase encoded by pncA within Mtb to the active moiety, pyrazinoic acid (POA) and PZA resistance is caused by loss-of-function mutations to pyrazinamidase. We have recently shown that POA induces targeted protein degradation of the enzyme PanD, a crucial component of the coenzyme A biosynthetic pathway essential in Mtb. Based on the newly identified mechanism of action of POA, along with the crystal structure of PanD bound to POA, we designed several POA analogs using structure for interpretation to improve potency and overcome PZA resistance. We prepared and tested ring and carboxylic acid bioisosteres as well as 3, 5, 6 substitutions on the ring to study the structure activity relationships of the POA scaffold. All the analogs were evaluated for their whole cell antimycobacterial activity, and a few representative molecules were evaluated for their binding affinity, towards PanD, through isothermal titration calorimetry. We report that analogs with ring and carboxylic acid bioisosteres did not significantly enhance the antimicrobial activity, whereas the alkylamino-group substitutions at the 3 and 5 position of POA were found to be up to 5 to 10-fold more potent than POA. Further development and mechanistic analysis of these analogs may lead to a next generation POA analog for treating TB.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Pirazinamida/farmacologia , Pirazinamida/metabolismo , Antituberculosos/farmacologia , Antituberculosos/metabolismo , Amidoidrolases/metabolismo , Tuberculose/microbiologia , Mutação , Relação Estrutura-Atividade , Ácidos Carboxílicos/metabolismo , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana
10.
Int J Mol Sci ; 23(18)2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36142891

RESUMO

The bile resistance of intestinal bacteria is among the key factors responsible for their successful colonization of and survival in the mammalian gastrointestinal tract. In this study, we demonstrated that lactate-producing Atopobiaceae bacteria (Leptogranulimonas caecicola TOC12T and Granulimonas faecalis OPF53T) isolated from mouse intestine showed high resistance to mammalian bile extracts, due to significant bile salt hydrolase (BSH) activity. We further succeeded in isolating BSH proteins (designated LcBSH and GfBSH) from L. caecicola TOC12T and G. faecalis OPF53T, respectively, and characterized their enzymatic features. Interestingly, recombinant LcBSH and GfBSH proteins exhibited BSH activity against 12 conjugated bile salts, indicating that LcBSH and GfBSH have much broader substrate specificity than the previously identified BSHs from lactic acid bacteria, which are generally known to hydrolyze six bile salt isomers. Phylogenetic analysis showed that LcBSH and GfBSH had no affinities with any known BSH subgroup and constituted a new BSH subgroup in the phylogeny. In summary, we discovered functional BSHs with broad substrate specificity from Atopobiaceae bacteria and demonstrated that these BSH enzymes confer bile resistance to L. caecicola TOC12T and G. faecalis OPF53T.


Assuntos
Actinobacteria , Lactobacillales , Actinobacteria/metabolismo , Amidoidrolases/metabolismo , Animais , Bile/metabolismo , Ácidos e Sais Biliares , Lactatos , Lactobacillales/metabolismo , Mamíferos/metabolismo , Camundongos , Filogenia , Especificidade por Substrato
11.
Int J Mol Sci ; 23(17)2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36077106

RESUMO

Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner. The purification of LapD reveals that it forms a complex with several proteins involved in LPS and phospholipid biosynthesis, including FtsH-LapA/B and Fab enzymes. Loss of LapD causes a reduction in LpxC amounts and vancomycin sensitivity, which can be restored by mutations that stabilize LpxC (mutations in lapB, ftsH and lpxC genes), revealing that LapD acts upstream of LapB-FtsH in regulating LpxC amounts. Interestingly, LapD absence results in the substantial retention of LPS in the inner membranes and synthetic lethality when either the lauroyl or the myristoyl acyl transferase is absent, which can be overcome by single-amino acid suppressor mutations in LPS flippase MsbA, suggesting LPS translocation defects in ΔlapD bacteria. Several genes whose products are involved in cell envelope homeostasis, including clsA, waaC, tig and micA, become essential in LapD's absence. Furthermore, the overproduction of acyl carrier protein AcpP or transcriptional factors DksA, SrrA can overcome certain defects of the LapD-lacking strain.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos , Oxirredutases/metabolismo , Aciltransferases/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Lipopolissacarídeos/metabolismo , Supressão Genética
12.
PLoS One ; 17(9): e0271540, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36048828

RESUMO

Human alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are members of the CREST superfamily of integral-membrane hydrolases. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. Although the structure of ACER3 was recently reported, catalytic roles for these residues have not been biochemically tested. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast mutant cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six-point mutants of the conserved CREST motif were developed that form a Zn-binding active site based on a recent crystal structure of human ACER3. Five point mutants completely lost their activity, with the exception of S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutant was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state. Together, these data indicate that ACER3 is a Zn2+-dependent amidase that catalyzes hydrolysis of ceramides via a similar mechanism to other soluble Zn-based amidases. Consistent with this notion, ACER3 was specifically inhibited by trichostatin A, a strong zinc chelator.


Assuntos
Ceramidase Alcalina , Ceramidas , Ceramidase Alcalina/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Ceramidases/metabolismo , Ceramidas/metabolismo , Humanos , Hidrólise , Zinco/metabolismo
13.
Curr Pharm Des ; 28(35): 2890-2900, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36154601

RESUMO

BACKGROUND: In bacteria, peptide deformylase (PDF), a metalloenzyme, removes N-formyl methionine from a nascent protein, which is a critical step in the protein maturation process. The enzyme is ubiquitously present in bacteria and possesses therapeutic target potential. Acarbose, an FDA-approved antidiabetic drug, is an alpha-glucosidase inhibitor of microbial origin. Clinical studies indicate that acarbose administration in humans can alter gut microbiota. As per the best of our knowledge, the antibacterial potential of acarbose has not been reported. OBJECTIVE: The present study aimed to check the binding ability of acarbose to the catalytic site of E. coli PDF and assess its in vitro antibacterial activity. METHODS: Molecular docking, molecular dynamic (MD) simulation, and MM-PBSA experiments were performed to study the binding potential of the catalytic site, and a disc diffusion assay was also employed to assess the antibacterial potential of acarbose. RESULTS: Acarbose was found to form a hydrogen bond and interact with the metal ion present at the catalytic site. The test compound showed a better docking score in comparison to the standard inhibitor of PDF. MD simulation results showed energetically stable acarbose-PDF complex formation in terms of RMSD, RMSF, Rg, SASA, and hydrogen bond formation throughout the simulation period compared to the actinonin-PDF complex. Furthermore, MM-PBSA calculations showed better binding free energy (ΔG) of acarbose PDF than the actinonin-PDF complex. Moreover, acarbose showed in vitro antibacterial activity. CONCLUSION: Acarbose forms conformational and thermodynamically stable interaction with the E. coli peptide deformylase catalytic site. Results of the present work necessitate in-depth antimicrobial potential studies on the effect of acarbose on drug resistance and nonresistant bacteria.


Assuntos
Acarbose , Escherichia coli , Humanos , Domínio Catalítico , Acarbose/farmacologia , Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Inibidores Enzimáticos/química , Amidoidrolases/química , Amidoidrolases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química
14.
Am J Physiol Regul Integr Comp Physiol ; 323(5): R749-R762, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36154489

RESUMO

The complexity of neuropathic pain and its associated comorbidities, including dysautonomia, make it difficult to treat. Overlap of anatomical regions and pharmacology of sympathosensory systems in the central nervous system (CNS) provide targets for novel treatment strategies. The dorsal periaqueductal gray (dPAG) is an integral component of both the descending pain modulation system and the acute stress response and is critically involved in both analgesia and the regulation of sympathetic activity. Local manipulation of the endocannabinoid signaling system holds great promise to provide analgesia without excessive adverse effects and also influence autonomic output. Inhibition of fatty acid amide hydrolase (FAAH) increases brain concentrations of the endocannabinoid N-arachidonoylethanolamine (AEA) and reduces pain-related behaviors in neuropathic pain models. Neuropathic hyperalgesia and reduced sympathetic tone are associated with increased FAAH activity in the dPAG, which suggests the hypothesis that inhibition of FAAH in the dPAG will normalize pain sensation and autonomic function in neuropathic pain. To test this hypothesis, the effects of systemic or intra-dPAG FAAH inhibition on hyperalgesia and dysautonomia developed after spared nerve injury (SNI) were assessed in male and female rats. Administration of the FAAH inhibitor PF-3845 into the dPAG reduces hyperalgesia behavior and the decrease in sympathetic tone induced by SNI. Prior administration of the CB1 receptor antagonist AM281, attenuated the antihyperalgesic and sympathetic effects of FAAH inhibition. No sex differences were identified. These data support an integrative role for AEA/CB1 receptor signaling in the dPAG contributing to the regulation of both hyperalgesia behavior and altered sympathetic tone in neuropathic pain.


Assuntos
Neuralgia , Disautonomias Primárias , Feminino , Masculino , Animais , Ratos , Endocanabinoides/farmacologia , Hiperalgesia/tratamento farmacológico , Substância Cinzenta Periaquedutal/metabolismo , Receptor CB1 de Canabinoide , Amidoidrolases/metabolismo , Neuralgia/tratamento farmacológico , Alcamidas Poli-Insaturadas/uso terapêutico
15.
Angew Chem Int Ed Engl ; 61(44): e202211774, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36083191

RESUMO

Obesity is a chronic health condition characterized by the accumulation of excessive body fat which can lead to and exacerbate cardiovascular disease, type-II diabetes, high blood pressure, and cancer through systemic inflammation. Unfortunately, visualizing key mediators of the inflammatory response, such as monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), in a selective manner is a profound challenge owing to an overlapping substrate scope that involves arachidonic acid (AA). Specifically, these enzymes work in concert to generate AA, which in the context of obesity, has been implicated to control appetite and energy metabolism. In this study, we developed the first selective activity-based sensing probes to detect MGL (PA-HD-MGL) and FAAH (PA-HD-FAAH) activity via photoacoustic imaging. Activation of PA-HD-MGL and PA-HD-FAAH by their target enzymes resulted in 1.74-fold and 1.59-fold signal enhancements, respectively. Due to their exceptional selectivity profiles and deep-tissue photoacoustic imaging capabilities, these probes were employed to measure MGL and FAAH activity in a murine model of obesity. Contrary to conflicting reports suggesting levels of MGL can be attenuated or elevated, our results support the latter. Indeed, we discovered a marked increase of both targets in the gastrointestinal tract. These key findings set the stage to uncover the role of the endocannabinoid pathway in obesity-mediated inflammation.


Assuntos
Endocanabinoides , Monoacilglicerol Lipases , Animais , Camundongos , Humanos , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Ácido Araquidônico , Modelos Animais de Doenças , Amidoidrolases/metabolismo , Obesidade/diagnóstico por imagem , Inflamação
16.
Microbiology (Reading) ; 168(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36129743

RESUMO

Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies. The ancient association between these two organisms accounts for many of their unique physiological adaptations. Similar to other obligate mutualists, Wigglesworthia's genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly's vertebrate blood-specific diet. These Wigglesworthia-derived B-vitamins play essential nutritional roles to maintain tsetse's physiological homeostasis as well as that of other members of the fly's microbiota. In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse's immune system during the larval period. Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia. These amidases in turn exhibit antiparasitic activity and decrease tsetse's ability to be colonized with parasitic trypanosomes, which reduce host fitness. Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.


Assuntos
Moscas Tsé-Tsé , Wigglesworthia , Amidoidrolases/metabolismo , Animais , Antiparasitários/metabolismo , Simbiose , Moscas Tsé-Tsé/parasitologia , Moscas Tsé-Tsé/fisiologia , Vitaminas/metabolismo , Wigglesworthia/metabolismo
17.
Rapid Commun Mass Spectrom ; 36(21): e9376, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-35945033

RESUMO

The analysis of glycoproteins and the comparison of protein N-glycosylation from different eukaryotic origins require unbiased and robust analytical workflows. The structural and functional analysis of vertebrate protein N-glycosylation currently depends extensively on bacterial peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidases (PNGases), which are indispensable enzymatic tools in releasing asparagine-linked oligosaccharides (N-glycans) from glycoproteins. So far, only limited PNGase candidates are available for N-glycans analysis, and particularly the analysis of plant and invertebrate N-glycans is hampered by the lack of suitable PNGases. Furthermore, liquid chromatography-mass spectrometry (LC-MS) workflows, such as hydrogen deuterium exchange mass spectrometry (HDX-MS), require a highly efficient enzymatic release of N-glycans at low pH values to facilitate the comprehensive structural analysis of glycoproteins. Herein, we describe a previously unstudied superacidic bacterial N-glycanase (PNGase H+ ) originating from the soil bacterium Rudaea cellulosilytica (Rc), which has significantly improved enzymatic properties compared to previously described PNGase H+ variants. Active and soluble recombinant PNGase Rc was expressed at a higher protein level (3.8-fold) and with higher specific activity (~56% increase) compared to the currently used PNGase H+ variant from Dyella japonicum (Dj). Recombinant PNGase Rc was able to deglycosylate the glycoproteins horseradish peroxidase and bovine lactoferrin significantly faster than PNGase Dj (10 min vs. 6 h). The versatility of PNGase Rc was demonstrated by releasing N-glycans from a diverse array of samples such as peach fruit, king trumpet mushroom, mouse serum, and the soil nematode Caenorhabditis elegans. The presence of only two disulfide bonds shown in the AlphaFold protein model (so far all other superacidic PNGases possess more disulfide bonds) could be corroborated by intact mass- and peptide mapping analysis and provides a possible explanation for the improved recombinant expression yield of PNGase Rc.


Assuntos
Asparagina , Espectrometria de Massa com Troca Hidrogênio-Deutério , Amidoidrolases/metabolismo , Animais , Medição da Troca de Deutério , Dissulfetos , Eucariotos/metabolismo , Gammaproteobacteria , Glicoproteínas/química , Peroxidase do Rábano Silvestre/metabolismo , Lactoferrina/metabolismo , Camundongos , Oligossacarídeos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/química , Solo
18.
PLoS Pathog ; 18(8): e1010750, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35930610

RESUMO

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.


Assuntos
Proteínas Periplásmicas , Repetições de Tetratricopeptídeos , Amidoidrolases/metabolismo , Biofilmes , Proteínas Periplásmicas/metabolismo , Polímeros
19.
Nat Commun ; 13(1): 4576, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35931690

RESUMO

Lipopolysaccharide (LPS) is an essential glycolipid and forms a protective permeability barrier for most Gram-negative bacteria. In E. coli, LPS levels are under feedback control, achieved by FtsH-mediated degradation of LpxC, which catalyzes the first committed step in LPS synthesis. FtsH is a membrane-bound AAA+ protease, and its protease activity toward LpxC is regulated by essential membrane proteins LapB and YejM. However, the regulatory mechanisms are elusive. We establish an in vitro assay to analyze the kinetics of LpxC degradation and demonstrate that LapB is an adaptor protein that utilizes its transmembrane helix to interact with FtsH and its cytoplasmic domains to recruit LpxC. Our YejM/LapB complex structure reveals that YejM is an anti-adaptor protein, competing with FtsH for LapB to inhibit LpxC degradation. Structural analysis unravels that LapB and LPS have overlapping binding sites in YejM. Thus, LPS levels control formation of the YejM/LapB complex to determine LpxC protein levels.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Amidoidrolases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
20.
Microbiol Spectr ; 10(4): e0220522, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35924842

RESUMO

Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. OTA detoxification strategies are significant to food safety. A superefficient enzyme ADH3 to OTA hydrolysis was isolated from the difunctional strain Stenotrophomonas sp. CW117 in our previous study. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA, which is an isoenzyme of ADH3. However, it is not as efficient a hydrolase as ADH3. The kinetic constant showed that the catalytic efficiency of ADH3 (Kcat/Km = 30,3938 s-1 · mM-1) against OTA was 29,113 times higher than that of NA (Kcat/Km = 10.4 s-1 · mM-1), indicating that ADH3 was the overwhelming superior detoxifying gene in CW117. Intriguingly, when gene na was knocked out from the CW117 genome, degradation activity of the Δna mutant was significantly reduced at the first 6 h, suggesting that the two enzymes might have an interactive effect on OTA transformation. Gene expressions and Western blotting assay showed that the Δna mutant and wild-type CW117 showed similar adh3 expression levels, but na deficiency decreased ADH3 protein level in CW117. Collectively, isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 but not as a dominant hydrolase for OTA transformation. IMPORTANCE Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. Previous OTA detoxification studies mainly focused on characterizations of degradation strains and detoxifying enzymes. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA from strain CW117, which is an isoenzyme of the efficient detoxifying enzyme ADH3. Isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 and, thus, enhanced the degradation activity of the strain. This is the first study on an isoenzyme improving the stability of another efficient detoxifying enzyme in vivo.


Assuntos
Micotoxinas , Ocratoxinas , Amidoidrolases/metabolismo , Aminoácidos/metabolismo , Aspergillus , Isoenzimas/metabolismo , Micotoxinas/metabolismo , Ocratoxinas/química , Ocratoxinas/metabolismo , Stenotrophomonas/metabolismo
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