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1.
Antonie Van Leeuwenhoek ; 117(1): 47, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38427176

RESUMO

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.


Assuntos
Álcool Desidrogenase , Metanol , Peptococcaceae , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Metanol/metabolismo , Oxirredução , Transferases/metabolismo , Sulfatos/metabolismo , Cobalto , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo
2.
Sci Rep ; 14(1): 5765, 2024 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459140

RESUMO

Autism spectrum disorder (ASD) is a complicated, lifelong neurodevelopmental disorder affecting verbal and non-verbal communication and social interactions. ASD signs and symptoms appear early in development before the age of 3 years. It is unlikely for a person to acquire autism after a period of normal development. However, we encountered an 8-year-old child who developed ASD later in life although his developmental milestones were normal at the beginning of life. Sequencing the complete coding part of the genome identified a hemizygous nonsense mutation (NM_001367857.2):c.1803C>G; (p.Tyr601Ter) in the gene (SATL1) encoding spermidine/spermine N1-acetyl transferase like 1. Screening an ASD cohort of 28 isolated patients for the SATL1 gene identified another patient with the same variant. Although SATL1 mutations have not been associated with any human diseases, our data suggests that a mutation in SATL1 is the underlying cause of ASD in our cases. In mammals, mutations in spermine synthase (SMS), an enzyme needed for the synthesis of spermidine polyamine, have been reported in a syndromic form of the X-linked mental retardation. Moreover, SATL1 gene expression studies showed a relatively higher expression of SATL1 transcripts in ASD related parts of the brain including the cerebellum, amygdala and frontal cortex. Additionally, spermidine has been characterized in the context of learning and memory and supplementations with spermidine increase neuroprotective effects and decrease age-induced memory impairment. Furthermore, spermidine biosynthesis is required for spontaneous axonal regeneration and prevents α-synuclein neurotoxicity in invertebrate models. Thus, we report, for the first time, that a mutation in the SATL1 gene could be a contributing factor in the development of autistic symptoms in our patients.


Assuntos
Transtorno do Espectro Autista , Espermidina , Animais , Criança , Humanos , Transtorno do Espectro Autista/genética , Poliaminas/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Transferases
3.
Int J Mol Sci ; 25(5)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38473957

RESUMO

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.


Assuntos
Acetatos , Carthamus tinctorius , Ciclopentanos , Oxilipinas , Transferases , Transferases/metabolismo , Ácido Clorogênico/metabolismo , Carthamus tinctorius/genética , Simulação de Acoplamento Molecular , Transcriptoma , Nucleotidiltransferases/metabolismo , Regulação da Expressão Gênica de Plantas
4.
Water Res ; 252: 121226, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38309071

RESUMO

The extensive exploration of antibiotic biodegradation by antibiotic-degrading bacteria in biological wastewater treatment processes has left a notable gap in understanding the behavior of these bacteria when exposed to antibiotics and the initiation of biodegradation processes. This study, therefore, delves into the adhesive behavior of Paraclostridium bifermentans, isolated from a bioreactor treating ciprofloxacin-laden wastewater, towards ciprofloxacin molecules. For the first time, this behavior is observed and characterized through quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy. The investigation further extends to identify key regulatory factors and mechanisms governing this adhesive behavior through a comparative proteomics analysis. The results reveal the dominance of extracellular proteins, particularly those involved in nucleotide binding, hydrolase, and transferase, in the adhesion process. These proteins play pivotal roles through direct chemical binding and the regulation of signaling molecule. Furthermore, QCM-D measurements provide evidence that transferase-related signaling molecules, especially tyrosine, augment the binding between ciprofloxacin and transferases, resulting in enhance ciprofloxacin removal by P. bifermentans (increased by ∼1.2-fold). This suggests a role for transferase-related signaling molecules in manipulating the adhesive behavior of P. bifermentans towards ciprofloxacin. These findings contribute to a new understanding of the prerequisites for antibiotic biodegradation and offer potential strategies for improving the application of antibiotic-degrading bacteria in the treatment of antibiotics-laden wastewater.


Assuntos
Antibacterianos , Ciprofloxacina , Antibacterianos/metabolismo , Ciprofloxacina/metabolismo , Águas Residuárias , Biodegradação Ambiental , Bactérias/metabolismo , Proteínas , Transferases/metabolismo
5.
Biochemistry ; 63(5): 671-687, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38393327

RESUMO

The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate (d-GAP). DXP is an essential bacteria-specific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of d-GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote LThDP activation. Here, we show that the substitution of putative network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. Substitutions predicted to disrupt the network have the effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predicted to strengthen the network fails to activate LThDP and has the effect to shift DXPS toward the closed state. Network-disrupting substitutions near the carboxylate of LThDP also have a pronounced effect to shift DXPS to an open state. These results offer initial insights to explain the long-lived LThDP intermediate and its activation through disruption of an active site network, which is unique to DXPS. These findings have important implications for DXPS function in bacteria and its development as an antibacterial target.


Assuntos
Difosfatos , Tiamina Pirofosfato , Domínio Catalítico , Tiamina Pirofosfato/metabolismo , Transferases/metabolismo , Ácido Pirúvico , Bactérias/metabolismo , Óxido Nítrico Sintase/metabolismo , Antibacterianos
6.
Proc Natl Acad Sci U S A ; 121(7): e2322375121, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38315835

RESUMO

Protein S-acyl transferases (PATs) catalyze S-acylation, a reversible post-translational modification critical for membrane association, trafficking, and stability of substrate proteins. Many plant proteins are potentially S-acylated but few have corresponding PATs identified. By using genomic editing, confocal imaging, pharmacological, genetic, and biochemical assays, we demonstrate that three Arabidopsis class C PATs positively regulate BR signaling through S-acylation of BRASSINOSTEROID-SIGNALING KINASE1 (BSK1). PAT19, PAT20, and PAT22 associate with the plasma membrane (PM) and the trans-Golgi network/early endosome (TGN/EE). Functional loss of all three genes results in a plethora of defects, indicative of reduced BR signaling and rescued by enhanced BR signaling. PAT19, PAT20, and PAT22 interact with BSK1 and are critical for the S-acylation of BSK1, and for BR signaling. The PM abundance of BSK1 was reduced by functional loss of PAT19, PAT20, and PAT22 whereas abolished by its S-acylation-deficient point mutations, suggesting a key role of S-acylation in its PM targeting. Finally, an active BR analog induces vacuolar trafficking and degradation of PAT19, PAT20, or PAT22, suggesting that the S-acylation of BSK1 by the three PATs serves as a negative feedback module in BR signaling.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Serina-Treonina Quinases , Acilação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Transferases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
7.
Biochemistry ; 63(5): 651-659, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38388156

RESUMO

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.


Assuntos
Nucleotídeos , Transferases , Humanos , Nucleotídeos/metabolismo , Transferases/metabolismo , Proteínas de Bactérias/química , Monofosfato de Adenosina/metabolismo , Processamento de Proteína Pós-Traducional
8.
Bone ; 181: 117043, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38341164

RESUMO

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.


Assuntos
Osteogênese , Fatores de Transcrição , Osteogênese/genética , Fatores de Transcrição/metabolismo , Lisina/metabolismo , Proteínas Nucleares/genética , Diferenciação Celular/genética , Epigênese Genética , Osteoblastos/metabolismo , Transferases/genética , Transferases/metabolismo
9.
Appl Environ Microbiol ; 90(3): e0207423, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38319094

RESUMO

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.


Assuntos
Bifidobacterium breve , Sistemas CRISPR-Cas , Humanos , Edição de Genes/métodos , Bifidobacterium breve/genética , Ácido Linoleico , Transferases/genética , Uracila
10.
Elife ; 122024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38358918

RESUMO

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.


Assuntos
Salmonella enterica , Transferases , Transferases/genética , Transferases/química , Antígenos O , Metabolismo dos Carboidratos , Membrana Celular , Salmonella enterica/genética
11.
ACS Chem Biol ; 19(3): 629-640, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38394345

RESUMO

Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1F383A. The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc- or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc- or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.


Assuntos
Glicoconjugados , Polissacarídeos , Humanos , Polissacarídeos/metabolismo , Membrana Celular/metabolismo , Transferases , Difosfato de Uridina
12.
J Am Chem Soc ; 146(5): 3220-3229, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38271668

RESUMO

Complex bacterial glycoconjugates drive interactions between pathogens, symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphosphosugar (UDP-sugar) substrate to a membrane-bound polyprenol-phosphate (Pren-P). The two distinct superfamilies of PGT enzymes (polytopic and monotopic) show striking differences in their structure and mechanism. We designed and synthesized a series of uridine bisphosphonates (UBPs), wherein the diphosphate of the UDP and UDP-sugar is replaced by a substituted methylene bisphosphonate (CXY-BPs; X/Y = F/F, Cl/Cl, (S)-H/F, (R)-H/F, H/H, CH3/CH3). UBPs and UBPs incorporating an N-acetylglucosamine (GlcNAc) substituent at the ß-phosphonate were evaluated as inhibitors of a polytopic PGT (WecA from Thermotoga maritima) and a monotopic PGT (PglC from Campylobacter jejuni). Although CHF-BP most closely mimics diphosphate with respect to its acid/base properties, the less basic CF2-BP conjugate more strongly inhibited PglC, whereas the more basic CH2-BP analogue was the strongest inhibitor of WecA. These surprising differences indicate different modes of ligand binding for the different PGT superfamilies, implicating a modified P-O- interaction with the structural Mg2+. For the monoPGT enzyme, the two diastereomeric CHF-BP conjugates, which feature a chiral center at the Pα-CHF-Pß carbon, also exhibited strikingly different binding affinities and the inclusion of GlcNAc with the native α-anomer configuration significantly improved binding affinity. UBP-sugars are thus revealed as informative new mechanistic probes of PGTs that may aid development of novel antibiotic agents for the exclusively prokaryotic monoPGT superfamily.


Assuntos
Difosfatos , Transferases , Humanos , Transferases/química , Uridina , Glicoconjugados/química , Difosfonatos , Açúcares , Difosfato de Uridina
13.
Ecotoxicol Environ Saf ; 271: 115954, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38232523

RESUMO

BACKGROUND: Nickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure. METHODS: We employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation. RESULT: Exposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, ß-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3ß/ß-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3ß/ß-Catenin pathway and c-Myc transcription in HBECs post nickel exposure. CONCLUSION: Our results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/ß-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients.


Assuntos
Níquel , beta Catenina , Humanos , Níquel/toxicidade , Níquel/metabolismo , beta Catenina/metabolismo , Sestrinas/metabolismo , Regulação para Cima , Transferases/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Epiteliais/metabolismo , Transformação Celular Neoplásica/genética , DNA/metabolismo , RNA Mensageiro/metabolismo , Ubiquitina Tiolesterase/metabolismo
14.
Eur J Pharmacol ; 967: 176355, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38280463

RESUMO

Pulmonary fibrosis is a challenging lung disease characterized by a bleak prognosis. A pivotal element in the progression of this disease is the dysregulated recruitment of macrophages. Nicotinamide phosphoribose transferase (NAMPT), secreted by alveolar epithelial cells and inflammatory cells, has been previously identified to influence macrophage inflammation in acute lung injury through the nicotinamide adenine dinucleotide (NAD) rescue synthesis pathway. Nonetheless, the exact role of NAMPT in the regulation of lung fibrosis is yet to be elucidated. In our research, we employed bleomycin (BLM) to induce pulmonary fibrosis in Namptflox/flox;Cx3cr1CreER mice, using Namptflox/flox mice as controls. Our findings revealed an augmented expression of NAMPT concurrent with a marked increase in the secretion of NAD and inflammatory cytokines such as IL-6, TNF-α, and TGF-ß1 post-BLM treatment. Furthermore, an upsurge in NAMPT-positive macrophages was observed in the lungs of BLM-treated Namptflox/flox mice. Notably, a conditional knockout of NAMPT (NAMPT cKO) in lung macrophages curtailed the BLM-induced inflammatory responses and significantly mitigated pulmonary fibrosis. This was associated with diminished phospho-Sirt1 (p-Sirt1) expression levels and a concomitant rise in mothers against decapentaplegic homolog 7 (Smad7) expression in BLM-treated mouse lungs and murine RAW 264.7 macrophage cells. Collectively, our data suggests that NAMPT exacerbates macrophage-driven inflammation and pulmonary fibrosis via the Sirt1-Smad7 pathway, positioning NAMPT as a promising therapeutic target for pulmonary fibrosis intervention.


Assuntos
Fibrose Pulmonar , Animais , Camundongos , Bleomicina/efeitos adversos , Citocinas/metabolismo , Inflamação , Macrófagos/metabolismo , NAD , Niacinamida , Nicotinamida Fosforribosiltransferase/genética , Fibrose Pulmonar/induzido quimicamente , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transferases
15.
Br J Cancer ; 130(6): 1059-1072, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38278976

RESUMO

BACKGROUND: Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors. METHODS: Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown. RESULTS: Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models. DISCUSSION: Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors.


Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Colorretais , Neoplasias Pulmonares , Neoplasias Pancreáticas , Humanos , Animais , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Transferases , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Mutação
16.
Plant Physiol Biochem ; 207: 108327, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38271860

RESUMO

Triclosan has been extensively used as a preservative in cosmetics and personal care products. However, its accumulation represents a real environmental threat. Thus, its phytotoxic impact needs more consideration. Our study was conducted to highlight the phytotoxic effect of triclosan on the growth, ROS homeostasis, and detoxification metabolism of two different plant species i.e., legumes (Glycine max) and grass (Avena sativa). Moreover, we investigated the potentiality of plant growth-promoting bacteria (ST-PGPB) in mitigating the phytotoxic effect of triclosan. Triclosan induced biomass (fresh and dry weights) reduction in both plants, but to a higher extent in oats. This decline was associated with a noticeable increment in the oxidative damage (e.g., MDA and H2O2) and detoxification metabolites such as metallothionein (MTC), phytochelatins (PCs), and glutathione-S-transferase (GST). This elevation was associated with a remarkable reduction in both enzymatic and non-enzymatic antioxidants. On the other hand, the bioactive strain of ST-PGPB, Salinicoccus sp. JzA1 significantly alleviated the harmful effect of triclosan on both soybean and oat plants by enhancing their biomass, photosynthesis, as well as levels of minerals (K, Ca, P, Mn, and Zn). In parallel, a striking quenching in oxidative damage and an obvious improvement in non-enzymatic (polyphenols, tocopherols, flavonoids) and enzymatic antioxidants were observed. Furthermore, Salinicoccus sp. JzA1 augmented the detoxification metabolism by enhancing the levels of phytochelatins, metallothionein, and glutathione-S-transferase (GST) activity in a species-specific manner which is more apparent in soybean rather than in oat plants. To this end, stress mitigating impact of Salinicoccus sp. JzA1 provides a basis to improve the resilience of crop species under cosmetics and personal care products toxicity.


Assuntos
Cosméticos , Triclosan , Avena/metabolismo , Triclosan/metabolismo , Triclosan/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Fitoquelatinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Antioxidantes/metabolismo , Estresse Oxidativo , Plantas/metabolismo , Homeostase , Cosméticos/metabolismo , Cosméticos/farmacologia , Metalotioneína/metabolismo , Transferases/metabolismo
17.
J Biomol Struct Dyn ; 42(2): 863-875, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37096664

RESUMO

Leishmaniasis is caused by Leishmania genus parasites and has a high mortality rate. The available drugs to treat leishmaniasis fail due to acquired resistance in parasites. Several enzymes of the Leishmania parasite have been used to design new therapeutic molecules against leishmaniasis. This study uses a pharmacophore-guided approach to design the drug candidate by targeting Leishmania N-Myristoyl transferase (LdNMT). From the initial sequence analysis of LdNMT, we have identified a unique 20 amino acid stretch exploited for screening and designing the small molecules. The pharmacophore for the myristate binding site on LdNMT was elucidated, and a heatmap was constructed. The leishmanial NMT pharmacophore has similarities with other pathogenic microorganisms. Moreover, substituting alanine in pharmacophoric residues elevates the affinity of myristate with NMT. Furthermore, a molecular dynamics (MD) simulation study was conducted to ascertain the stability of the mutants and or wild type. The wild-type NMT has a comparatively low affinity to myristate compared to alanine mutants, indicating that hydrophobic residues favor the myristate binding. The molecules were initially designed by using pharmacophore as a sieving mechanism. In subsequent steps, the selected molecules screened against leishmanial unique amino acid stretch and subsequently with human, leishmanial full-size NMTs. The compounds BP5, TYI, DMU, 3PE and 4UL were the top hits and chemical features similar to the myristate. The molecule 4UL was found to be highly specific towards leishmanial NMT over human NMT, suggesting the molecule is a strong leishmanial NMT inhibitor. The molecule can be taken further to assess it in in-vitro conditions.


Assuntos
Leishmania , Leishmaniose , Humanos , Transferases , Farmacóforo , Miristatos , Leishmaniose/tratamento farmacológico , Desenho de Fármacos , Alanina , Aminoácidos
18.
J Biomol Struct Dyn ; 42(4): 1711-1724, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37325855

RESUMO

Zika virus (ZIKV) spread is considered a major public health threat by the World Health Organization (WHO). There are no vaccines or drugs available to control the infection of the Zika virus, therefore a highly effective medicinal molecule is urgently required. In this study, a computationally intensive investigation was performed to identify a potent natural compound that could inhibit the ZIKV NS5 methyltransferase. This research approach is based on target-based drug identification principles where the native inhibitor SAH (S-adenosylhomocysteine) of ZIKV NS5 methyltransferase was selected as a reference. High-throughput virtual screening and tanimoto similarity coefficient were applied to the natural compound library for ranking the potential candidates. The top five compounds were selected for interaction analysis, MD simulation, total binding free energy through MM/GBSA, and steered MD simulation. Among these compounds, Adenosine 5'-monophosphate monohydrate, Tubercidin, and 5-Iodotubercidin showed stable binding to the protein compared to the native compound, SAH. These three compounds also showed less fluctuations in RMSF in contrast to native compound. Additionally, the same interacting residues observed in SAH also made strong interactions with these three compounds. Adenosine 5'-monophosphate monohydrate and 5-Iodotubercidin had greater total binding free energies than the reference ligand. Moreover, the dissociation resistance of all three compounds was equivalent to that of the reference ligand. This study suggested binding properties of three-hit compounds that could be used to develop drugs against Zika virus infections.Communicated by Ramaswamy H. Sarma.


Assuntos
Infecção por Zika virus , Zika virus , Humanos , Simulação de Dinâmica Molecular , Ligantes , Proteínas não Estruturais Virais/química , Adenosina , Metiltransferases/química , Transferases/metabolismo , Transferases/farmacologia , Simulação de Acoplamento Molecular , Antivirais/farmacologia , Antivirais/química
19.
Vet Microbiol ; 288: 109932, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38043447

RESUMO

Bovine herpesvirus 1(BoHV-1) is an important bovine pathogen that causes great economic loss to cattle farms worldwide. The virus-productive infection in bovine kidney (MDBK) cells results in ATP depletion. The mechanisms are not well understood. Mitochondrial fatty acid ß-oxidation (FAO) is an important energy source in many tissues with high energy demand. Since carnitine palmitoyl-transferase 1 A (CPT1A) is the rate-limiting enzyme of FAO, we investigated the interactions between virus-productive infection and CPT1A signaling. Here, we found that virus-productive infection at the later stage significantly decreased CPT1A protein levels in all the detected cells, including MDBK, A549, and Neuro-2A cells, differentially altered the accumulation of CPT1A proteins in the nucleus and cytosol, and re-localized the protein in the nucleus. Etomoxir (ETO), an irreversible inhibitor of CPT1A, inhibited viral replication and partially interfered with the ability of BoHV-1 to alter CPT1A accumulation in the nucleus but not in the cytosol. Furthermore, ETO consistently reduced RNA levels of two viral regulatory proteins (bICP0 and bICP22) and protein expression of virion-associated proteins during productive infection, further supporting the important roles of CPT1A signaling in BoHV-1 productive infection. These data, for the first time, suggest that CPT1A is potentially involved in BoHV-1 productive infection.


Assuntos
Doenças dos Bovinos , Infecções por Herpesviridae , Herpesvirus Bovino 1 , Bovinos , Animais , Herpesvirus Bovino 1/genética , Replicação Viral , Infecções por Herpesviridae/veterinária , Transferases/metabolismo , Carnitina/metabolismo
20.
Cell Death Differ ; 31(2): 170-187, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38062245

RESUMO

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.


Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Humanos , Camundongos , Proteínas de Ciclo Celular , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Proteômica , Fatores de Transcrição/genética , Transferases , Proteína GLI1 em Dedos de Zinco/genética
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