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1.
Proc Natl Acad Sci U S A ; 121(25): e2321890121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38857388

RESUMO

In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal α-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Estresse Oxidativo , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Fosforilação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Tirosina Quinases/metabolismo , Peróxido de Hidrogênio/metabolismo , Amidoidrolases/metabolismo
2.
Cell Death Dis ; 15(6): 399, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849335

RESUMO

The loss of dopaminergic neurons in the substantia nigra is a hallmark of pathology in Parkinson's disease (PD). Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the critical enzyme responsible for the degradation of asymmetric dimethylarginine (ADMA) which inhibits nitric oxide (NO) synthase and has been implicated in neurodegeneration. Mitochondrial dysfunction, particularly in the mitochondria-associated endoplasmic reticulum membrane (MAM), plays a critical role in this process, although the specific molecular target has not yet been determined. This study aims to examine the involvement of DDAH-1 in the nigrostriatal dopaminergic pathway and PD pathogenesis. The distribution of DDAH-1 in the brain and its colocalization with dopaminergic neurons were observed. The loss of dopaminergic neurons and aggravated locomotor disability after rotenone (ROT) injection were showed in the DDAH-1 knockout rat. L-arginine (ARG) and NO donors were employed to elucidate the role of NO respectively. In vitro, we investigated the effects of DDAH-1 knockdown or overexpression on cell viability and mitochondrial functions, as well as modulation of ADMA/NO levels using ADMA or ARG. MAM formation was assessed by the Mitofusin2 oligomerization and the mitochondrial ubiquitin ligase (MITOL) phosphorylation. We found that DDAH-1 downregulation resulted in enhanced cell death and mitochondrial dysfunctions, accompanied by elevated ADMA and reduced NO levels. However, the recovered NO level after the ARG supplement failed to exhibit a protective effect on mitochondrial functions and partially restored cell viability. DDAH-1 overexpression prevented ROT toxicity, while ADMA treatment attenuated these protective effects. The declines of MAM formation in ROT-treated cells were exacerbated by DDAH-1 downregulation via reduced MITOL phosphorylation, which was reversed by DDAH-1 overexpression. Together, the abundant expression of DDAH-1 in nigral dopaminergic neurons may exert neuroprotective effects by maintaining MAM formation and mitochondrial function probably via ADMA, indicating the therapeutic potential of targeting DDAH-1 for PD.


Assuntos
Amidoidrolases , Arginina , Neurônios Dopaminérgicos , Retículo Endoplasmático , Mitocôndrias , Óxido Nítrico , Doença de Parkinson , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Animais , Amidoidrolases/metabolismo , Amidoidrolases/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/genética , Arginina/metabolismo , Arginina/análogos & derivados , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Ratos , Óxido Nítrico/metabolismo , Masculino , Ratos Sprague-Dawley , Humanos , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Rotenona/farmacologia , Proteínas Mitocondriais/metabolismo , Membranas Associadas à Mitocôndria
3.
Protein Sci ; 33(7): e5067, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38864716

RESUMO

The N-degron pathway determines the half-life of proteins by selectively destabilizing the proteins bearing N-degrons. N-terminal glutamine amidohydrolase 1 (NTAQ1) plays an essential role in the arginine N-degron (Arg/N-degron) pathway as an initializing enzyme via the deamidation of the N-terminal (Nt) glutamine (Gln). However, the Nt-serine-bound conformation of hNTAQ1 according to the previously identified crystal structure suggests the possibility of other factors influencing the recognition of Nt residues by hNTAQ1. Hence, in the current study, we aimed to further elucidate the substrate recognition of hNTAQ1; specifically, we explored 12 different substrate-binding conformations of hNTAQ1 depending on the subsequent residue of Nt-Gln. Results revealed that hNTAQ1 primarily interacts with the protein Nt backbone, instead of the side chain, for substrate recognition. Here, we report that the Nt backbone of proteins appears to be a key component of hNTAQ1 function and is the main determinant of substrate recognition. Moreover, not all second residues from Nt-Gln, but rather distinctive and charged residues, appeared to aid in detecting substrate recognition. These new findings define the substrate-recognition process of hNTAQ1 and emphasize the importance of the subsequent Gln residue in the Nt-Gln degradation system. Our extensive structural and biochemical analyses provide insights into the substrate specificity of the N-degron pathway and shed light on the mechanism underlying hNTAQ1 substrate recognition. An improved understanding of the protein degradation machinery could aid in developing therapies to promote overall health through enhanced protein regulation, such as targeted protein therapies.


Assuntos
Arginina , Humanos , Especificidade por Substrato , Arginina/química , Arginina/metabolismo , Modelos Moleculares , Glutamina/metabolismo , Glutamina/química , Amidoidrolases/química , Amidoidrolases/metabolismo , Amidoidrolases/genética , Conformação Proteica , Proteólise , Degrons
4.
Artigo em Inglês | MEDLINE | ID: mdl-38765527

RESUMO

Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.


Assuntos
Amidoidrolases , Arginina , Haplótipos , Polimorfismo Genético , Pré-Eclâmpsia , Humanos , Feminino , Amidoidrolases/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/sangue , Gravidez , Adulto , Arginina/análogos & derivados , Arginina/sangue , Arginina/genética , Adulto Jovem
5.
Biomacromolecules ; 25(6): 3607-3619, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38776179

RESUMO

Studying how synthetic polymer assemblies respond to sequential enzymatic stimuli can uncover intricate interactions in biological systems. Using amidase- and esterase-responsive PEG-based diblock (DBA) and triblock amphiphiles (TBAs), we created two distinct formulations: amidase-responsive DBA with esterase-responsive TBA and vice versa. We studied their cascade responses to the two enzymes and the sequence of their introduction. These formulations underwent cascade mesophase transitions upon the addition of the DBA-degrading enzyme, transitioning from (i) coassembled micelles to (ii) triblock-based hydrogel, and ultimately to (iii) dissolved polymers when exposed to the TBA hydrolyzing enzyme. The specific pathway of the two mesophase transitions depended on the compositions of the formulations and the enzyme introduction sequence. The results highlight the potential for designing polymeric formulations with programmable multistep enzymatic responses, mimicking the complex behavior of biological macromolecules.


Assuntos
Polietilenoglicóis , Polietilenoglicóis/química , Micelas , Esterases/química , Esterases/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Transição de Fase , Polímeros/química , Hidrogéis/química
6.
Int J Biol Macromol ; 270(Pt 2): 132281, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38740150

RESUMO

DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.


Assuntos
Proteínas de Bactérias , Enterococcus faecium , Enterococcus faecium/enzimologia , Especificidade por Substrato , Ligantes , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Conformação Proteica , Zinco/química , Zinco/metabolismo , Domínio Catalítico , Amidoidrolases/química , Amidoidrolases/metabolismo , Amidoidrolases/genética , Cristalografia por Raios X , Sequência de Aminoácidos , Ligação Proteica
7.
Biomed Pharmacother ; 175: 116677, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38701570

RESUMO

The current pharmacological approaches to multiple sclerosis (MS) target its inflammatory and autoimmune components, but effective treatments to foster remyelination and axonal repair are still lacking. We therefore selected two targets known to be involved in MS pathogenesis: N-acylethanolamine-hydrolyzing acid amidase (NAAA) and glycogen synthase kinase-3ß (GSK-3ß). We tested whether inhibiting these targets exerted a therapeutic effect against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The combined inhibition of NAAA and GSK-3ß by two selected small-molecule compounds, ARN16186 (an NAAA inhibitor) and AF3581 (a GSK-3ß inhibitor), effectively mitigated disease progression, rescuing the animals from paralysis and preventing a worsening of the pathology. The complementary activity of the two inhibitors reduced the infiltration of immune cells into the spinal cord and led to the formation of thin myelin sheaths around the axons post-demyelination. Specifically, the inhibition of NAAA and GSK-3ß modulated the over-activation of NF-kB and STAT3 transcription factors in the EAE-affected mice and induced the nuclear translocation of ß-catenin, reducing the inflammatory insult and promoting the remyelination process. Overall, this work demonstrates that the dual-targeting of key aspects responsible for MS progression could be an innovative pharmacological approach to tackle the pathology.


Assuntos
Amidoidrolases , Encefalomielite Autoimune Experimental , Glicogênio Sintase Quinase 3 beta , Camundongos Endogâmicos C57BL , Esclerose Múltipla , Animais , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Camundongos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Feminino , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , NF-kappa B/metabolismo , Inibidores Enzimáticos/farmacologia , Bainha de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos
8.
Mol Genet Metab ; 142(2): 108489, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38718669

RESUMO

BACKGROUND: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. METHODS: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. RESULTS: Measuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6-13.6 µmol/L [n = 45] vs 0.44; 0.24-0.99 µmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1-9.9 µmol/L [n = 8]) and neonatal controls (0.93; 0.4-1.8 µmol/L [n = 784]) (p < 0.05). CONCLUSIONS: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.


Assuntos
Ácido Aspártico , Doença de Canavan , Teste em Amostras de Sangue Seco , Triagem Neonatal , Espectrometria de Massas em Tandem , Humanos , Doença de Canavan/diagnóstico , Doença de Canavan/sangue , Doença de Canavan/genética , Recém-Nascido , Triagem Neonatal/métodos , Teste em Amostras de Sangue Seco/métodos , Espectrometria de Massas em Tandem/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/sangue , Cromatografia Líquida , Feminino , Masculino , Lactente , Pré-Escolar , Espectrometria de Massa com Cromatografia Líquida , Amidoidrolases
9.
J Hazard Mater ; 473: 134716, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38797074

RESUMO

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 µg/L OTA was completely degraded by 1.0 µg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-ß-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.


Assuntos
Lysobacter , Ocratoxinas , Ocratoxinas/metabolismo , Ocratoxinas/toxicidade , Lysobacter/metabolismo , Lysobacter/genética , Amidoidrolases/metabolismo , Amidoidrolases/genética , Carboxipeptidases/metabolismo , Carboxipeptidases/genética , Hidrolases/metabolismo , Hidrolases/genética
10.
mBio ; 15(6): e0094324, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38727244

RESUMO

Bile acids play a critical role in the emulsification of dietary lipids, a critical step in the primary function of the small intestine, which is the digestion and absorption of food. Primary bile acids delivered into the small intestine are conjugated to enhance functionality, in part, by increasing aqueous solubility and preventing passive diffusion of bile acids out of the gut lumen. Bile acid function can be disrupted by the gut microbiota via the deconjugation of primary bile acids by bile salt hydrolases (BSHs), leading to their conversion into secondary bile acids through the expression of bacterial bile acid-inducible genes, a process often observed in malabsorption due to small intestinal bacterial overgrowth. By modeling the small intestinal microbiota in vitro using human small intestinal ileostomy effluent as the inocula, we show here that the infusion of physiologically relevant levels of oxygen, normally found in the proximal small intestine, reduced deconjugation of primary bile acids, in part, through the expansion of bacterial taxa known to have a low abundance of BSHs. Further recapitulating the small intestinal bile acid composition of the small intestine, limited conversion of primary into secondary bile acids was observed. Remarkably, these effects were preserved among four separate communities, each inoculated with a different small intestinal microbiota, despite a high degree of taxonomic variability under both anoxic and aerobic conditions. In total, these results provide evidence for a previously unrecognized role that the oxygenated environment of the small intestine plays in the maintenance of normal digestive physiology. IMPORTANCE: Conjugated primary bile acids are produced by the liver and exist at high concentrations in the proximal small intestine, where they are critical for proper digestion. Deconjugation of these bile acids with subsequent transformation via dehydroxylation into secondary bile acids is regulated by the colonic gut microbiota and reduces their digestive function. Using an in vitro platform modeling the small intestinal microbiota, we analyzed the ability of this community to transform primary bile acids and studied the effect of physiological levels of oxygen normally found in the proximal small intestine (5%) on this metabolic process. We found that oxygenation of the small intestinal microbiota inhibited the deconjugation of primary bile acids in vitro. These findings suggest that luminal oxygen levels normally found in the small intestine may maintain the optimal role of bile acids in the digestive process by regulating bile acid conversion by the gut microbiota.


Assuntos
Ácidos e Sais Biliares , Microbioma Gastrointestinal , Intestino Delgado , Oxigênio , Ácidos e Sais Biliares/metabolismo , Humanos , Intestino Delgado/microbiologia , Intestino Delgado/metabolismo , Oxigênio/metabolismo , Bactérias/metabolismo , Bactérias/genética , Bactérias/classificação , Amidoidrolases
11.
Food Res Int ; 187: 114409, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38763660

RESUMO

Ochratoxin A (OTA) is a notorious mycotoxin commonly contaminating food products worldwide. In this study, an OTA-degrading strain Brevundimonas diminuta HAU429 was isolated by using hippuryl-L-phenylalanine as the sole carbon source. The biodegradation of OTA by strain HAU429 was a synergistic effect of intracellular and extracellular enzymes, which transformed OTA into ochratoxin α (OTα) through peptide bond cleavage. Cytotoxicity tests and cell metabolomics confirmed that the transformation of OTA into OTα resulted in the detoxification of its hepatotoxicity since OTA but not OTα disturbed redox homeostasis and induced oxidative damage to hepatocytes. Genome mining identified nine OTA hydrolase candidates in strain HAU429. They were heterologously expressed in Escherichia coli, and three novel amidohydrolase BT6, BT7 and BT9 were found to display OTA-hydrolyzing activity. BT6, BT7 and BT9 showed less than 45 % sequence identity with previously identified OTA-degrading amidohydrolases. BT6 and BT7 shared 60.9 % amino acid sequence identity, and exhibited much higher activity towards OTA than BT9. BT6 and BT7 could completely degrade 1 µg mL-1 of OTA within 1 h and 50 min, while BT9 hydrolyzed 100 % of OTA in the reaction mixture by 12 h. BT6 was the most thermostable retaining 38 % of activity after incubation at 70 °C for 10 min, while BT7 displayed the highest tolerance to ethanal remaining 76 % of activity in the presence of 6 % ethanol. This study could provide new insights towards microbial OTA degradation and promote the development of enzyme-catalyzed OTA detoxification during food processing.


Assuntos
Caulobacteraceae , Ocratoxinas , Ocratoxinas/metabolismo , Ocratoxinas/toxicidade , Caulobacteraceae/metabolismo , Caulobacteraceae/genética , Biodegradação Ambiental , Amidoidrolases/metabolismo , Amidoidrolases/genética , Contaminação de Alimentos
12.
J Appl Microbiol ; 135(5)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38724455

RESUMO

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.


Assuntos
Quitina , Quitina/metabolismo , Amidoidrolases/metabolismo , Amidoidrolases/genética , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Gammaproteobacteria/enzimologia , Gammaproteobacteria/metabolismo
13.
Biochemistry ; 63(10): 1322-1334, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38696389

RESUMO

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.


Assuntos
Amidas , Proteínas Periplásmicas de Ligação , Amidas/metabolismo , Amidas/química , Cristalografia por Raios X , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Aminoácidos/metabolismo , Mesorhizobium/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Modelos Moleculares , Amidoidrolases/metabolismo , Amidoidrolases/química , Cálcio/metabolismo , Ligação Proteica
14.
Acta Biochim Pol ; 71: 12299, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38721302

RESUMO

This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.


Assuntos
Amidoidrolases , Escherichia coli , Mutagênese , Amidoidrolases/genética , Amidoidrolases/metabolismo , Amidoidrolases/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Sequência de Aminoácidos , Mutação
15.
Nat Commun ; 15(1): 4026, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740822

RESUMO

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.


Assuntos
Amidoidrolases , Doença de Canavan , Proteólise , Humanos , Amidoidrolases/genética , Amidoidrolases/metabolismo , Doença de Canavan/genética , Doença de Canavan/metabolismo , Células HEK293 , Substituição de Aminoácidos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Ubiquitina/metabolismo , Termodinâmica
16.
Cells ; 13(10)2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38786051

RESUMO

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.


Assuntos
Amidoidrolases , Peptídeo Relacionado com Gene de Calcitonina , Hiperalgesia , Gânglio Trigeminal , Animais , Masculino , Hiperalgesia/tratamento farmacológico , Ratos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Amidoidrolases/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/sangue , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Ratos Sprague-Dawley , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Endocanabinoides/metabolismo , Nitroglicerina/farmacologia , Modelos Animais de Doenças , Citocinas/metabolismo , Citocinas/sangue , Transtornos de Enxaqueca/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Oligopeptídeos , Proteínas e Peptídeos Salivares
17.
Mar Drugs ; 22(5)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38786603

RESUMO

Naturally occurring echinocandin B and FR901379 are potent antifungal lipopeptides featuring a cyclic hexapeptide nucleus and a fatty acid side chain. They are the parent compounds of echinocandin drugs for the treatment of severe fungal infections caused by the Candida and Aspergilla species. To minimize hemolytic toxicity, the native fatty acid side chains in these drug molecules are replaced with designer acyl side chains. The deacylation of the N-acyl side chain is, therefore, a crucial step for the development and manufacturing of echinocandin-type antibiotics. Echinocandin E (ECE) is a novel echinocandin congener with enhanced stability generated via the engineering of the biosynthetic machinery of echinocandin B (ECB). In the present study, we report the discovery of the first echinocandin E acylase (ECEA) using the enzyme similarity tool (EST) for enzymatic function mining across protein families. ECEA is derived from Streptomyces sp. SY1965 isolated from a sediment collected from the Mariana Trench. It was cloned and heterologously expressed in S. lividans TK24. The resultant TKecea66 strain showed efficient cleavage activity of the acyl side chain of ECE, showing promising applications in the development of novel echinocandin-type therapeutics. Our results also provide a showcase for harnessing the essentially untapped biodiversity from the hadal ecosystems for the discovery of functional molecules.


Assuntos
Antifúngicos , Equinocandinas , Streptomyces , Streptomyces/enzimologia , Streptomyces/genética , Equinocandinas/química , Antifúngicos/farmacologia , Antifúngicos/química , Amidoidrolases/metabolismo , Proteínas Fúngicas
18.
Toxins (Basel) ; 16(5)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38787069

RESUMO

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.


Assuntos
Aflatoxinas , Amidoidrolases , Aspergillus flavus , Aspergillus flavus/genética , Aspergillus flavus/enzimologia , Aspergillus flavus/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Aflatoxinas/biossíntese , Aflatoxinas/metabolismo , Aflatoxinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Quitina/metabolismo , Parede Celular/metabolismo
19.
Sci Rep ; 14(1): 11587, 2024 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773239

RESUMO

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.


Assuntos
Amidoidrolases , Clonagem Molecular , Eucommiaceae , Regulação da Expressão Gênica de Plantas , Nicotiana , Plantas Geneticamente Modificadas , Eucommiaceae/genética , Eucommiaceae/metabolismo , Plantas Geneticamente Modificadas/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Nicotiana/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Filogenia , Sequência de Aminoácidos , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Oxilipinas/farmacologia , Oxilipinas/metabolismo
20.
Sci Rep ; 14(1): 11103, 2024 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750093

RESUMO

Safe and effective pain management is a critical healthcare and societal need. The potential for acute liver injury from paracetamol (ApAP) overdose; nephrotoxicity and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use; and opioids' addiction are unresolved challenges. We developed SRP-001, a non-opioid and non-hepatotoxic small molecule that, unlike ApAP, does not produce the hepatotoxic metabolite N-acetyl-p-benzoquinone-imine (NAPQI) and preserves hepatic tight junction integrity at high doses. CD-1 mice exposed to SRP-001 showed no mortality, unlike a 70% mortality observed with increasing equimolar doses of ApAP within 72 h. SRP-001 and ApAP have comparable antinociceptive effects, including the complete Freund's adjuvant-induced inflammatory von Frey model. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception region, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways/networks, including endocannabinoid signaling, genes pertaining to mechanical nociception, and fatty acid amide hydrolase (FAAH). Both regulate the expression of key genes encoding FAAH, 2-arachidonoylglycerol (2-AG), cannabinoid receptor 1 (CNR1), CNR2, transient receptor potential vanilloid type 4 (TRPV4), and voltage-gated Ca2+ channel. Phase 1 trial (NCT05484414) (02/08/2022) demonstrates SRP-001's safety, tolerability, and favorable pharmacokinetics, including a half-life from 4.9 to 9.8 h. Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.


Assuntos
Acetaminofen , Analgésicos , Ácidos Araquidônicos , Substância Cinzenta Periaquedutal , Transcriptoma , Animais , Masculino , Camundongos , Acetaminofen/efeitos adversos , Amidoidrolases/metabolismo , Amidoidrolases/genética , Analgésicos/farmacologia , Ácidos Araquidônicos/farmacologia , Benzoquinonas/farmacologia , Glicerídeos , Substância Cinzenta Periaquedutal/metabolismo , Substância Cinzenta Periaquedutal/efeitos dos fármacos
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