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1.
Molecules ; 29(11)2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38893310

RESUMO

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.


Assuntos
Arildialquilfosfatase , Proteômica , Arildialquilfosfatase/metabolismo , Arildialquilfosfatase/química , Humanos , Proteômica/métodos , Redobramento de Proteína , Pseudomonas aeruginosa/enzimologia , Estabilidade Enzimática , Biofilmes , Processamento de Proteína Pós-Traducional
2.
Molecules ; 29(11)2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38893400

RESUMO

The outbreak of SARS-CoV-2, also known as the COVID-19 pandemic, is still a critical risk factor for both human life and the global economy. Although, several promising therapies have been introduced in the literature to inhibit SARS-CoV-2, most of them are synthetic drugs that may have some adverse effects on the human body. Therefore, the main objective of this study was to carry out an in-silico investigation into the medicinal properties of Petiveria alliacea L. (P. alliacea L.)-mediated phytocompounds for the treatment of SARS-CoV-2 infections since phytochemicals have fewer adverse effects compared to synthetic drugs. To explore potential phytocompounds from P. alliacea L. as candidate drug molecules, we selected the infection-causing main protease (Mpro) of SARS-CoV-2 as the receptor protein. The molecular docking analysis of these receptor proteins with the different phytocompounds of P. alliacea L. was performed using AutoDock Vina. Then, we selected the three top-ranked phytocompounds (myricitrin, engeletin, and astilbin) as the candidate drug molecules based on their highest binding affinity scores of -8.9, -8.7 and -8.3 (Kcal/mol), respectively. Then, a 100 ns molecular dynamics (MD) simulation study was performed for their complexes with Mpro using YASARA software, computed RMSD, RMSF, PCA, DCCM, MM/PBSA, and free energy landscape (FEL), and found their almost stable binding performance. In addition, biological activity, ADME/T, DFT, and drug-likeness analyses exhibited the suitable pharmacokinetics properties of the selected phytocompounds. Therefore, the results of this study might be a useful resource for formulating a safe treatment plan for SARS-CoV-2 infections after experimental validation in wet-lab and clinical trials.


Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Fitoquímicos , SARS-CoV-2 , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/química , Antivirais/farmacologia , Antivirais/química , Antivirais/uso terapêutico , Humanos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/uso terapêutico , COVID-19/virologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico
3.
Molecules ; 29(11)2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38893578

RESUMO

BACKGROUND: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract. METHODS: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site. RESULTS: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies. CONCLUSIONS: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.


Assuntos
Antocianinas , Antivirais , Mirtilos Azuis (Planta) , Proteases 3C de Coronavírus , Extratos Vegetais , Inibidores de Proteases , SARS-CoV-2 , Mirtilos Azuis (Planta)/química , Antocianinas/farmacologia , Antocianinas/química , Antivirais/farmacologia , Antivirais/química , Chlorocebus aethiops , Células Vero , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Tratamento Farmacológico da COVID-19 , Humanos , Simulação de Acoplamento Molecular , COVID-19/virologia , Glucosídeos
4.
Physiol Plant ; 176(3): e14380, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38894644

RESUMO

Phototropism movement is crucial for plants to adapt to various environmental changes. Plant P-type H+-ATPase (HA) plays diverse roles in signal transduction during cell expansion, regulation of cellular osmotic potential and stomatal opening, and circadian movement. Despite numerous studies on the genome-wide analysis of Vitis vinifera, no research has been done on the P-type H+-ATPase family genes, especially concerning pulvinus-driven leaf movement. In this study, 55 VvHAs were identified and classified into nine distinct subgroups (1 to 9). Gene members within the same subgroups exhibit similar features in motif, intron/exon, and protein tertiary structures. Furthermore, four pairs of genes were derived by segmental duplication in grapes. Cis-acting element analysis identified numerous light/circadian-related elements in the promoters of VvHAs. qRT-PCR analysis showed that several genes of subgroup 7 were highly expressed in leaves and pulvinus during leaf movement, especially VvHA14, VvHA15, VvHA16, VvHA19, VvHA51, VvHA52, and VvHA54. Additionally, we also found that the VvHAs genes were asymmetrically expressed on both sides of the extensor and flexor cell of the motor organ, the pulvinus. The expression of VvHAs family genes in extensor cells was significantly higher than that in flexor cells. Overall, this study serves as a foundation for further investigations into the functions of VvHAs and contributes to the complex mechanisms underlying grapevine pulvinus growth and development.


Assuntos
Regulação da Expressão Gênica de Plantas , Fototropismo , Folhas de Planta , Proteínas de Plantas , ATPases Translocadoras de Prótons , Vitis , Vitis/genética , Vitis/fisiologia , Vitis/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fototropismo/genética , Fototropismo/fisiologia , Pulvínulo/genética , Pulvínulo/metabolismo , Pulvínulo/fisiologia , Membrana Celular/metabolismo , Filogenia , Família Multigênica
5.
Protein Sci ; 33(7): e5075, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38895978

RESUMO

Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.


Assuntos
Piruvato Quinase , Zymomonas , Humanos , Zymomonas/enzimologia , Zymomonas/genética , Zymomonas/química , Zymomonas/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Substituição de Aminoácidos , Estabilidade Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Estabilidade Enzimática , Fígado/enzimologia , Fígado/metabolismo , Fígado/química , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato/química
6.
Protein Sci ; 33(7): e5071, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38895984

RESUMO

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.


Assuntos
Mycobacterium tuberculosis , NAD , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/química , NAD/metabolismo , Domínios Proteicos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Cristalografia por Raios X , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/química , Evolução Molecular , Modelos Moleculares , Filogenia , NAD+ Nucleosidase/metabolismo , NAD+ Nucleosidase/química , NAD+ Nucleosidase/genética
7.
Protein Sci ; 33(7): e5080, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38896002

RESUMO

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.


Assuntos
Protease de HIV , Transcriptase Reversa do HIV , HIV-1 , Multimerização Proteica , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Transcriptase Reversa do HIV/genética , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/química , Humanos , Modelos Moleculares , Dimerização
8.
Yi Chuan ; 46(6): 502-508, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38886153

RESUMO

Ssu72 is a component of the yeast cleavage/polyadenylation factor (CPF) complex, which catalyzes the dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II at S5-P and S7-P. It has been shown that Ssu72 phosphatase is involved in regulating chromosome cohesion during mitosis. To further clarify whether Ssu72 phosphatase affects chromosome separation during meiotic division in Schizosaccharomyces pombe, we utilized green fluorescent protein (GFP) to label centromeres and red fluorescent protein to label microtubule protein Atb2. The entire meiotic chromosome separation process of ssu72∆ cells was observed in real-time under fluorescence microscope. It was found that two spindles of ssu72∆ cells crossed during the metaphase and anaphase of the second meiotic division, and this spindle crossing led to a new type of spore defect distribution pattern. The results of this study can provide important reference significance for studying the roles of phosphatase Ssu72 in higher organisms.


Assuntos
Meiose , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Fuso Acromático , Schizosaccharomyces/genética , Schizosaccharomyces/enzimologia , Fuso Acromático/genética , Fuso Acromático/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Segregação de Cromossomos
9.
Nat Commun ; 15(1): 5167, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886362

RESUMO

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.


Assuntos
Chaetomium , Metilenotetra-Hidrofolato Redutase (NADPH2) , S-Adenosilmetionina , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Regulação Alostérica , Chaetomium/enzimologia , Chaetomium/metabolismo , Chaetomium/genética , Fosforilação , Humanos , Cristalografia por Raios X , Modelos Moleculares , Flavina-Adenina Dinucleotídeo/metabolismo , Flavina-Adenina Dinucleotídeo/química
10.
Sci Rep ; 14(1): 13917, 2024 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886497

RESUMO

Chinese rose (Rosa chinensis) is an important ornamental plant, with economic, cultural, and symbolic significance. During the application of outdoor greening, adverse environments such as high temperature and drought are often encountered, which affect its application scope and ornamental quality. The starch phosphorylase (Pho) gene family participate in the synthesis and decomposition of starch, not only related to plant energy metabolism, but also plays an important role in plant stress resistance. The role of Pho in combating salinity and high temperature stress in R. chinensis remains unknown. In this work, 4 Phos from R. chinensis were detected with Pfam number of Pho (PF00343.23) and predicted by homolog-based prediction (HBP). The Phos are characterized by sequence lengths of 821 to 997 bp, and the proteins are predicted to subcellularly located in the plastid and cytoplasm. The regulatory regions of the Phos contain abundant stress and phytohormone-responsive cis-acting elements. Based on transcriptome analysis, the Phos were found to respond to abiotic stress factors such as drought, salinity, high temperature, and plant phytohormone of jasmonic acid and salicylic acid. The response of Phos to abiotic stress factors such as salinity and high temperature was confirmed by qRT-PCR analysis. To evaluate the genetic characteristics of Phos, a total of 69 Phos from 17 species were analyzed and then classified into 3 groups in phylogenetic tree. The collinearity analysis of Phos in R. chinensis and other species was conducted for the first time. This work provides a view of evolution for the Pho gene family and indicates that Phos play an important role in abiotic stress response of R. chinensis.


Assuntos
Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Rosa , Amido Fosforilase , Estresse Fisiológico , Estresse Fisiológico/genética , Rosa/genética , Rosa/enzimologia , Rosa/metabolismo , Amido Fosforilase/genética , Amido Fosforilase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilação da Expressão Gênica , Secas , Genoma de Planta , Salinidade
11.
CNS Neurosci Ther ; 30(6): e14808, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38887205

RESUMO

OBJECTIVE: Phenylethanolamine N-methyltransferase (PNMT)-expressing neurons in the nucleus tractus solitarii (NTS) contribute to the regulation of autonomic functions. However, the neural circuits linking these neurons to other brain regions remain unclear. This study aims to investigate the connectivity mechanisms of the PNMT-expressing neurons in the NTS (NTSPNMT neurons). METHODS: The methodologies employed in this study included a modified rabies virus-based retrograde neural tracing technique, conventional viral anterograde tracing, and immunohistochemical staining procedures. RESULTS: A total of 43 upstream nuclei projecting to NTSPNMT neurons were identified, spanning several key brain regions including the medulla oblongata, pons, midbrain, cerebellum, diencephalon, and telencephalon. Notably, dense projections to the NTSPNMT neurons were observed from the central amygdaloid nucleus, paraventricular nucleus of the hypothalamus, area postrema, and the gigantocellular reticular nucleus. In contrast, the ventrolateral medulla, lateral parabrachial nucleus, and lateral hypothalamic area were identified as the primary destinations for axon terminals originating from NTSPNMT neurons. Additionally, reciprocal projections were evident among 21 nuclei, primarily situated within the medulla oblongata. CONCLUSION: Our research findings demonstrate that NTSPNMT neurons form extensive connections with numerous nuclei, emphasizing their essential role in the homeostatic regulation of vital autonomic functions.


Assuntos
Neurônios , Feniletanolamina N-Metiltransferase , Núcleo Solitário , Animais , Feniletanolamina N-Metiltransferase/metabolismo , Feniletanolamina N-Metiltransferase/genética , Núcleo Solitário/enzimologia , Núcleo Solitário/metabolismo , Núcleo Solitário/citologia , Neurônios/metabolismo , Neurônios/enzimologia , Masculino , Vias Eferentes/enzimologia , Vias Aferentes/enzimologia , Ratos Sprague-Dawley , Mapeamento Encefálico/métodos , Ratos
12.
Sci Rep ; 14(1): 14015, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890382

RESUMO

Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.


Assuntos
Aspergillus niger , Galactanos , Mananas , Oligossacarídeos , Gomas Vegetais , Prebióticos , beta-Manosidase , Mananas/química , Mananas/metabolismo , Gomas Vegetais/química , Galactanos/química , Aspergillus niger/enzimologia , Oligossacarídeos/química , Hidrólise , beta-Manosidase/metabolismo , beta-Manosidase/química , Concentração de Íons de Hidrogênio , Ácidos Graxos Voláteis/metabolismo , Difração de Raios X , Temperatura , Lactobacillus/metabolismo , Probióticos
13.
Commun Biol ; 7(1): 742, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890421

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS.


Assuntos
Quinase do Linfoma Anaplásico , Antimaláricos , Lisina-tRNA Ligase , Plasmodium falciparum , Inibidores de Proteínas Quinases , Plasmodium falciparum/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Lisina-tRNA Ligase/antagonistas & inibidores , Lisina-tRNA Ligase/metabolismo , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/genética , Antimaláricos/farmacologia , Antimaláricos/química , Relação Estrutura-Atividade , Humanos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
14.
BMC Microbiol ; 24(1): 216, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38890647

RESUMO

OBJECTIVE: This study aims to conduct an in-depth genomic analysis of a carbapenem-resistant Proteus mirabilis strain to uncover the distribution and mechanisms of its resistance genes. METHODS: The research primarily utilized whole-genome sequencing to analyze the genome of the Proteus mirabilis strain. Additionally, antibiotic susceptibility tests were conducted to evaluate the strain's sensitivity to various antibiotics, and related case information was collected to analyze the clinical distribution characteristics of the resistant strain. RESULTS: Study on bacterial strain WF3430 from a tetanus and pneumonia patient reveals resistance to multiple antibiotics due to extensive use. Whole-genome sequencing exposes a 4,045,480 bp chromosome carrying 29 antibiotic resistance genes. Two multidrug-resistant (MDR) gene regions, resembling Tn6577 and Tn6589, were identified (MDR Region 1: 64.83 Kb, MDR Region 2: 85.64 Kbp). These regions, consist of integrative and conjugative elements (ICE) structures, highlight the intricate multidrug resistance in clinical settings. CONCLUSION: This study found that a CR-PMI strain exhibits a unique mechanism for acquiring antimicrobial resistance genes, such as blaNDM-1, located on the chromosome instead of plasmids. According to the results, there is increasing complexity in the mechanisms of horizontal transmission of resistance, necessitating a comprehensive understanding and implementation of targeted control measures in both hospital and community settings.


Assuntos
Antibacterianos , Proteínas de Bactérias , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Infecções por Proteus , Proteus mirabilis , Sequenciamento Completo do Genoma , beta-Lactamases , Proteus mirabilis/genética , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/enzimologia , Proteus mirabilis/isolamento & purificação , beta-Lactamases/genética , Humanos , Farmacorresistência Bacteriana Múltipla/genética , Antibacterianos/farmacologia , Infecções por Proteus/microbiologia , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Genoma Bacteriano/genética , Carbapenêmicos/farmacologia
15.
Commun Biol ; 7(1): 725, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38867087

RESUMO

The rising use of plastic results in an appalling amount of waste which is scattered into the environment. One of these plastics is PET which is mainly used for bottles. We have identified and characterized an esterase from Streptomyces, annotated as LipA, which can efficiently degrade the PET-derived oligomer BHET. The Streptomyces coelicolor ScLipA enzyme exhibits varying sequence similarity to several BHETase/PETase enzymes, including IsPETase, TfCut2, LCC, PET40 and PET46. Of 96 Streptomyces strains, 18% were able to degrade BHET via one of three variants of LipA, named ScLipA, S2LipA and S92LipA. SclipA was deleted from S. coelicolor resulting in reduced BHET degradation. Overexpression of all LipA variants significantly enhanced BHET degradation. All variants were expressed in E. coli for purification and biochemical analysis. The optimum conditions were determined as pH 7 and 25 °C for all variants. The activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and indents on the surface of PET films, comparable to the activity of previously described TfCut2 under the same conditions. The abundance of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more polar substrates including these polluting plastics.


Assuntos
Streptomyces , Streptomyces/enzimologia , Streptomyces/genética , Microbiologia do Solo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Biodegradação Ambiental , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética , Esterases/metabolismo , Esterases/genética , Esterases/química , Polietilenotereftalatos/metabolismo
16.
BMC Genomics ; 25(1): 593, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38867153

RESUMO

BACKGROUND: Terpenes are important components of plant aromas, and terpene synthases (TPSs) are the key enzymes driving terpene diversification. In this study, we characterized the volatile terpenes in five different Chrysanthemum nankingense tissues. In addition, genome-wide identification and expression analysis of TPS genes was conducted utilizing an improved chromosome-scale genome assembly and tissue-specific transcriptomes. The biochemical functions of three representative TPSs were also investigated. RESULTS: We identified tissue-specific volatile organic compound (VOC) and volatile terpene profiles. The improved Chrysanthemum nankingense genome assembly was high-quality, including a larger assembled size (3.26 Gb) and a better contig N50 length (3.18 Mb) compared to the old version. A total of 140 CnTPS genes were identified, with the majority representing the TPS-a and TPS-b subfamilies. The chromosomal distribution of these TPS genes was uneven, and 26 genes were included in biosynthetic gene clusters. Closely-related Chrysanthemum taxa were also found to contain diverse TPS genes, and the expression profiles of most CnTPSs were tissue-specific. The three investigated CnTPS enzymes exhibited versatile activities, suggesting multifunctionality. CONCLUSIONS: We systematically characterized the structure and diversity of TPS genes across the Chrysanthemum nankingense genome, as well as the potential biochemical functions of representative genes. Our results provide a basis for future studies of terpene biosynthesis in chrysanthemums, as well as for the breeding of improved chrysanthemum varieties.


Assuntos
Alquil e Aril Transferases , Chrysanthemum , Genoma de Planta , Família Multigênica , Terpenos , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Chrysanthemum/genética , Chrysanthemum/enzimologia , Terpenos/metabolismo , Filogenia , Compostos Orgânicos Voláteis/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcriptoma
17.
Microb Cell Fact ; 23(1): 170, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38867249

RESUMO

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.


Assuntos
Bacillus subtilis , Peso Molecular , Paenibacillus , beta-Galactosidase , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , beta-Galactosidase/metabolismo , beta-Galactosidase/genética , Paenibacillus/enzimologia , Paenibacillus/genética , Citoplasma/metabolismo , Regiões Promotoras Genéticas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Sinais Direcionadores de Proteínas
18.
Cardiovasc Diabetol ; 23(1): 202, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38867293

RESUMO

The specific pathophysiological pathways through which diabetes exacerbates myocardial ischemia/reperfusion (I/R) injury remain unclear; however, dysregulation of immune and inflammatory cells, potentially driven by abnormalities in their number and function due to diabetes, may play a significant role. In the present investigation, we simulated myocardial I/R injury by inducing ischemia through ligation of the left anterior descending coronary artery in mice for 40 min, followed by reperfusion for 24 h. Previous studies have indicated that protein kinase Cß (PKCß) is upregulated under hyperglycemic conditions and is implicated in the development of various diabetic complications. The Y4 RNA fragment is identified as the predominant small RNA component present in the extracellular vesicles of cardio sphere-derived cells (CDCs), exhibiting notable anti-inflammatory properties in the contexts of myocardial infarction and cardiac hypertrophy. Our investigation revealed that the administration of Y4 RNA into the ventricular cavity of db/db mice following myocardial I/R injury markedly enhanced cardiac function. Furthermore, Y4 RNA was observed to facilitate M2 macrophage polarization and interleukin-10 secretion through the suppression of PKCß activation. The mechanism by which Y4 RNA affects PKCß by regulating macrophage activation within the inflammatory environment involves the inhibition of ERK1/2 phosphorylation In our study, the role of PKCß in regulating macrophage polarization during myocardial I/R injury was investigated through the use of PKCß knockout mice. Our findings indicate that PKCß plays a crucial role in modulating the inflammatory response associated with macrophage activation in db/db mice experiencing myocardial I/R, with a notable exacerbation of this response observed upon significant upregulation of PKCß expression. In vitro studies further elucidated the protective mechanism by which Y4 RNA modulates the PKCß/ERK1/2 signaling pathway to induce M2 macrophage activation. Overall, our findings suggest that Y4 RNA plays an anti-inflammatory role in diabetic I/R injury, suggesting a novel therapeutic approach for managing myocardial I/R injury in diabetic individuals.


Assuntos
Modelos Animais de Doenças , Macrófagos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica , Proteína Quinase C beta , Transdução de Sinais , Animais , Proteína Quinase C beta/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/genética , Macrófagos/metabolismo , Macrófagos/enzimologia , Masculino , Interleucina-10/metabolismo , Interleucina-10/genética , Camundongos , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/fisiopatologia , Células Cultivadas , Fenótipo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ativação de Macrófagos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Função Ventricular Esquerda , Fosforilação
19.
Gigascience ; 132024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38869150

RESUMO

Viral helicases are promising targets for the development of antiviral therapies. Given their vital function of unwinding double-stranded nucleic acids, inhibiting them blocks the viral replication cycle. Previous studies have elucidated key structural details of these helicases, including the location of substrate binding sites, flexible domains, and the discovery of potential inhibitors. Here we present a series of new Galaxy tools and workflows for performing and analyzing molecular dynamics simulations of viral helicases. We first validate them by demonstrating recapitulation of data from previous simulations of Zika (NS3) and SARS-CoV-2 (NSP13) helicases in apo and complex with inhibitors. We further demonstrate the utility and generalizability of these Galaxy workflows by applying them to new cases, proving their usefulness as a widely accessible method for exploring antiviral activity.


Assuntos
Simulação de Dinâmica Molecular , SARS-CoV-2 , SARS-CoV-2/enzimologia , Zika virus/enzimologia , Fluxo de Trabalho , RNA Helicases/química , RNA Helicases/metabolismo , Humanos , DNA Helicases/química , DNA Helicases/metabolismo , Antivirais/química , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Sítios de Ligação , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
20.
Eur J Sport Sci ; 24(6): 824-833, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38874978

RESUMO

We investigated the associations of low handgrip strength (HGS, i.e., a marker of muscular fitness) with liver fat content (LFC) and serum liver enzymes in a population-based setting. We used data from 2700 participants (51.7% women), aged 21-90 years, from two independent cohorts of the population-based Study of Health in Pomerania (SHIP-START-2 and SHIP-TREND-0). Cross-sectional, multivariable adjusted regression models were performed to examine the associations of HGS with LFC, measured by magnetic resonance imaging and serum liver enzymes. We found significant inverse associations of HGS with both LFC and serum liver enzymes. Specifically, a 10-kg lower HGS was associated with a 0.59% (95% confidence interval [CI]: 0.24-0.94; p = 0.001) higher LFC, a 0.051 µkatal/L (95% CI: 0.005-0.097; p = 0.031) higher gamma-glutamyltransferase (GGT) concentration and a 0.010 µkatal/L (95% CI: 0.001-0.020; p = 0.023) higher aspartate aminotransferase (AST) concentration. The adjusted odds-ratio for prevalent hepatic steatosis (defined by a MRI-PDFF ≥5.1%) per 10-kg lower HGS was 1.21 (95% CI: 1.04-1.40; p = 0.014). When considering only obese individuals, those with low HGS had a 1.58% (95% CI: 0.18-2.98; p = 0.027) higher mean LFC and higher chance of prevalent hepatic steatosis (adjusted OR 1.74, 95% CI: 1.15-2.62; p = 0.009) compared to individuals with high HGS. We found similar associations in individuals with overweight, but not in those with normal weight. Lower HGS was strongly associated with both higher LFC and higher serum GGT and AST concentrations. Future studies might clarify whether these findings reflect adverse effects of a sedentary lifestyle or aging on the liver.


Assuntos
Aspartato Aminotransferases , Força da Mão , Fígado , gama-Glutamiltransferase , Humanos , Pessoa de Meia-Idade , Feminino , Masculino , Adulto , Idoso , Estudos Transversais , Aspartato Aminotransferases/sangue , Fígado/enzimologia , Idoso de 80 Anos ou mais , gama-Glutamiltransferase/sangue , Adulto Jovem , Alemanha/epidemiologia , Imageamento por Ressonância Magnética , Comportamento Sedentário , Fígado Gorduroso/sangue , Alanina Transaminase/sangue
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