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1.
FASEB J ; 38(11): e23702, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38837439

RESUMO

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.


Assuntos
Diferenciação Celular , Histonas , Mioblastos , Piruvato Quinase , Animais , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Camundongos , Fosforilação , Histonas/metabolismo , Histonas/genética , Mioblastos/metabolismo , Mioblastos/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a Hormônio da Tireoide , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Isoenzimas/metabolismo , Isoenzimas/genética
3.
Biol Pharm Bull ; 47(6): 1136-1143, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38866522

RESUMO

Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.


Assuntos
Proteínas de Transferência de Fosfolipídeos , Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Células HEK293 , Esfingomielinas/metabolismo , Esfingomielinas/biossíntese , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Isoenzimas/metabolismo , Isoenzimas/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/enzimologia
4.
Carbohydr Res ; 541: 109169, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38838492

RESUMO

It is well established that tumour cells undergo metabolic changes to acquire biological advantage over normal cells with activation of the glycolytic pathway, a process termed "Warburg effect". Enzyme isoforms are alternative enzymatic forms with the same function but with different biochemical or epigenetic features. Moreover, isoforms may have varying impacts on different metabolic pathways. We challenge ourselves to analyse the glycolytic and gluconeogenic enzymes and isoforms in breast cancer, a complex and heterogeneous pathology, associated with high incidence and mortality rates especially among women. We analysed epithelial and tumour cell lines by RT-PCR and compared values to a publicly available database for the expression profile of normal and tumour tissues (Gepia) of enzymes and enzymatic isoforms from glycolytic and gluconeogenic pathways. Additionally, GeneMANIA was used to evaluate interactions, pathways, and attributes of each glycolytic/gluconeogenic steps. The findings reveal that the enzymes and enzymatic isoforms expressed in cell culture were somewhat different from those in breast tissue. We propose that the tumor microenvironment plays a crucial role in the expression of glycolytic and gluconeogenic enzymes and isoforms in tumour cells. Nonetheless, they not only participate in glycolytic and gluconeogenic enzymatic activities but may also influence other pathways, such as the Pentose-Phosphate-Pathway, TCA cycle, as well as other carbohydrate, lipid, and amino acid metabolism.


Assuntos
Neoplasias da Mama , Gluconeogênese , Glicólise , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/enzimologia , Feminino , Linhagem Celular Tumoral , Isoenzimas/metabolismo , Isoenzimas/genética
5.
Protein J ; 43(3): 592-602, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38733555

RESUMO

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).


Assuntos
IMP Desidrogenase , IMP Desidrogenase/metabolismo , IMP Desidrogenase/química , IMP Desidrogenase/genética , Animais , Camundongos , Isoenzimas/metabolismo , Isoenzimas/química , Isoenzimas/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Retina/metabolismo , Retina/enzimologia , Ligação Proteica , Humanos
6.
Chemosphere ; 358: 142249, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38705405

RESUMO

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs' mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.


Assuntos
Clorofenóis , Glucuronosiltransferase , Microssomos Hepáticos , Glucuronosiltransferase/metabolismo , Clorofenóis/metabolismo , Animais , Microssomos Hepáticos/metabolismo , Humanos , Ratos , Poluentes Ambientais/metabolismo , Isoenzimas/metabolismo , Glucuronídeos/metabolismo
7.
Cancer Immunol Immunother ; 73(7): 127, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38739169

RESUMO

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.


Assuntos
Carcinoma Hepatocelular , DNA Metiltransferase 3A , Epigênese Genética , L-Lactato Desidrogenase , Neoplasias Hepáticas , Microambiente Tumoral , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Microambiente Tumoral/imunologia , Humanos , Animais , Camundongos , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , DNA Metiltransferase 3A/metabolismo , Regulação Neoplásica da Expressão Gênica , Metilação de DNA , Isoenzimas/genética , Isoenzimas/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Prognóstico
8.
CNS Neurosci Ther ; 30(5): e14741, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38702940

RESUMO

AIMS: Despite the success of single-cell RNA sequencing in identifying cellular heterogeneity in ischemic stroke, clarifying the mechanisms underlying these associations of differently expressed genes remains challenging. Several studies that integrate gene expression and gene expression quantitative trait loci (eQTLs) with genome wide-association study (GWAS) data to determine their causal role have been proposed. METHODS: Here, we combined Mendelian randomization (MR) framework and single cell (sc) RNA sequencing to study how differently expressed genes (DEGs) mediating the effect of gene expression on ischemic stroke. The hub gene was further validated in the in vitro model. RESULTS: We identified 2339 DEGs in 10 cell clusters. Among these DEGs, 58 genes were associated with the risk of ischemic stroke. After external validation with eQTL dataset, lactate dehydrogenase B (LDHB) is identified to be positively associated with ischemic stroke. The expression of LDHB has also been validated in sc RNA-seq with dominant expression in microglia and astrocytes, and melatonin is able to reduce the LDHB expression and activity in vitro ischemic models. CONCLUSION: Our study identifies LDHB as a novel biomarker for ischemic stroke via combining the sc RNA-seq and MR analysis.


Assuntos
AVC Isquêmico , L-Lactato Desidrogenase , Melatonina , Análise da Randomização Mendeliana , Análise de Sequência de RNA , Animais , Humanos , Estudo de Associação Genômica Ampla/métodos , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Análise da Randomização Mendeliana/métodos , Locos de Características Quantitativas , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Camundongos
9.
J Biol Chem ; 300(5): 107278, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38599380

RESUMO

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.


Assuntos
Glucuronosiltransferase , Isoenzimas , Microssomos Hepáticos , Proteínas Recombinantes , Animais , Humanos , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/química , Isoenzimas/metabolismo , Isoenzimas/genética , Cinética , Microssomos Hepáticos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Magnetismo , Microssomos/química , Microssomos/metabolismo
10.
Plant Physiol Biochem ; 210: 108631, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38657550

RESUMO

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Germinação , Glutamato-Amônia Ligase , Sementes , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/enzimologia , Sementes/crescimento & desenvolvimento , Sementes/genética , Sementes/enzimologia , Germinação/genética , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas , Isoenzimas/genética , Isoenzimas/metabolismo
11.
Pflugers Arch ; 476(7): 1041-1064, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38658400

RESUMO

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCß. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCß do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.


Assuntos
Cricetulus , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Proteína Quinase C , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Animais , Fosforilação , Células HEK293 , Humanos , Ratos , Proteína Quinase C/metabolismo , Células CHO , Receptores Adrenérgicos alfa 1/metabolismo , Miócitos Cardíacos/metabolismo , Masculino , Ratos Wistar , Proteína Quinase C-alfa/metabolismo , Isoenzimas/metabolismo
12.
Cell Rep ; 43(4): 114047, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38607916

RESUMO

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina , L-Lactato Desidrogenase , Ácido Láctico , Humanos , Células Secretoras de Insulina/metabolismo , Animais , L-Lactato Desidrogenase/metabolismo , Camundongos , Ácido Láctico/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Isoenzimas/metabolismo , Ciclo do Ácido Cítrico , Camundongos Endogâmicos C57BL , Masculino
13.
J Phys Chem B ; 128(14): 3383-3397, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38563384

RESUMO

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.


Assuntos
Histidina , Poliquetos , Animais , Histidina/química , Isoenzimas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hemoglobinas/química , Peroxidases/química , Peroxidase/química , Poliquetos/química , Poliquetos/metabolismo , Cristalografia por Raios X
14.
Sci Rep ; 14(1): 8050, 2024 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580665

RESUMO

Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a) has been reported to be highly selective to sulfate pregnenolone. This study aimed to clarify the effect of missense single nucleotide polymorphisms (SNPs) of the human SULT2B1 gene on the sulfating activity of coded SULT2B1a allozymes toward Pregnenolone. To investigate the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of pregnenolone by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were generated, expressed, and purified using established procedures. Human SULT2B1a SNPs were identified by a comprehensive database search. 13 SULT2B1a nonsynonymous missense coding SNPs (cSNPs) were selected, and site-directed mutagenesis was used to generate the corresponding cDNAs, packaged in pGEX-2TK expression vector, encoding these 13 SULT2B1a allozymes, which were bacterially expressed in BL21 E. coli cells and purified by glutathione-Sepharose affinity chromatography. Purified SULT2B1a allozymes were analyzed for sulfating activities towards pregnenolone. In comparison with the wild-type SULT2B1a, of the 13 allozymes, 11 showed reduced activity toward pregnenolone at 0.1 µM. Specifically, P134L and R259Q allozymes, reported to be involved in autosomal-recessive congenital ichthyosis, displayed low activity (1-10%) toward pregnenolone. The findings of this study may demonstrate the impact of genetic polymorphism on the sulfation of pregnenolone in individuals with different SULT2B1 genotypes.


Assuntos
Isoenzimas , Pregnenolona , Humanos , Isoenzimas/metabolismo , Escherichia coli/metabolismo , Sulfotransferases/metabolismo , Polimorfismo de Nucleotídeo Único
15.
Cells ; 13(8)2024 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-38667330

RESUMO

BACKGROUND: Gaucher disease (GD) is caused by glucocerebrosidase (GCase) enzyme deficiency, leading to glycosylceramide (Gb-1) and glucosylsphingosine (Lyso-Gb-1) accumulation. The pathological hallmark for GD is an accumulation of large macrophages called Gaucher cells (GCs) in the liver, spleen, and bone marrow, which are associated with chronic organ enlargement, bone manifestations, and inflammation. Tartrate-resistant acid phosphatase type 5 (TRAP5 protein, ACP5 gene) has long been a nonspecific biomarker of macrophage/GCs activation; however, the discovery of two isoforms of TRAP5 has expanded its significance. The discovery of TRAP5's two isoforms revealed that it is more than just a biomarker of macrophage activity. While TRAP5a is highly expressed in macrophages, TRAP5b is secreted by osteoclasts. Recently, we have shown that the elevation of TRAP5b in plasma is associated with osteoporosis in GD. However, the role of TRAP isoforms in GD and how the accumulation of Gb-1 and Lyso-Gb-1 affects TRAP expression is unknown. METHODS: 39 patients with GD were categorized into cohorts based on bone mineral density (BMD). TRAP5a and TRAP5b plasma levels were quantified by ELISA. ACP5 mRNA was estimated using RT-PCR. RESULTS: An increase in TRAP5b was associated with reduced BMD and correlated with Lyso-Gb-1 and immune activator chemokine ligand 18 (CCL18). In contrast, the elevation of TRAP5a correlated with chitotriosidase activity in GD. Lyso-Gb-1 and plasma seemed to influence the expression of ACP5 in macrophages. CONCLUSIONS: As an early indicator of BMD alteration, measurement of circulating TRAP5b is a valuable tool for assessing osteopenia-osteoporosis in GD, while TRAP5a serves as a biomarker of macrophage activation in GD. Understanding the distinct expression pattern of TRAP5 isoforms offers valuable insight into both bone disease and the broader implications for immune system activation in GD.


Assuntos
Doença de Gaucher , Isoformas de Proteínas , Fosfatase Ácida Resistente a Tartarato , Doença de Gaucher/metabolismo , Doença de Gaucher/genética , Humanos , Fosfatase Ácida Resistente a Tartarato/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Densidade Óssea , Macrófagos/metabolismo , Biomarcadores/metabolismo , Biomarcadores/sangue , Isoenzimas/metabolismo , Isoenzimas/genética
16.
Ecotoxicol Environ Saf ; 276: 116281, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38581907

RESUMO

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.


Assuntos
Glucuronídeos , Glucuronosiltransferase , Microssomos Hepáticos , Fenóis , Glucuronosiltransferase/metabolismo , Humanos , Animais , Fenóis/toxicidade , Fenóis/metabolismo , Glucuronídeos/metabolismo , Poluentes Ambientais/toxicidade , Poluentes Ambientais/metabolismo , Cães , Ratos , Isoenzimas/metabolismo , Especificidade da Espécie
17.
Biochem J ; 481(9): 601-613, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38592741

RESUMO

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.


Assuntos
Músculo Esquelético , Óxido Nítrico Sintase Tipo I , Animais , Masculino , Camundongos , Éxons , Isoenzimas/metabolismo , Isoenzimas/genética , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo I/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Biochemistry ; 63(9): 1194-1205, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38598309

RESUMO

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.


Assuntos
Hordeum , Hordeum/enzimologia , Hordeum/genética , Especificidade por Substrato , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Glucanos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/química , Mutagênese , beta-Glucanas/metabolismo
19.
Cancer Lett ; 590: 216869, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38593918

RESUMO

Lysine acetyltransferase 7 (KAT7), a histone acetyltransferase, has recently been identified as an oncoprotein and has been implicated in the development of various malignancies. However, its specific role in head and neck squamous carcinoma (HNSCC) has not been fully elucidated. Our study revealed that high expression of KAT7 in HNSCC patients is associated with poor survival prognosis and silencing KAT7 inhibits the Warburg effect, leading to reduced proliferation, invasion, and metastatic potential of HNSCC. Further investigation uncovered a link between the high expression of KAT7 in HNSCC and tumor-specific glycolytic metabolism. Notably, KAT7 positively regulates Lactate dehydrogenase A (LDHA), a key enzyme in metabolism, to promote lactate production and create a conducive environment for tumor proliferation and metastasis. Additionally, KAT7 enhances LDHA activity and upregulates LDHA protein expression by acetylating the lysine 118 site of LDHA. Treatment with WM3835, a KAT7 inhibitor, effectively suppressed the growth of subcutaneously implanted HNSCC cells in mice. In conclusion, our findings suggest that KAT7 exerts pro-cancer effects in HNSCC by acetylating LDHA and may serve as a potential therapeutic target. Inhibiting KAT7 or LDHA expression holds promise as a therapeutic strategy to suppress the growth and progression of HNSCC.


Assuntos
Proliferação de Células , Neoplasias de Cabeça e Pescoço , Histona Acetiltransferases , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Animais , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Acetilação , Linhagem Celular Tumoral , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Camundongos , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Lisina Acetiltransferases/metabolismo , Lisina Acetiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Efeito Warburg em Oncologia , Masculino , Feminino , Movimento Celular , Ensaios Antitumorais Modelo de Xenoenxerto , Invasividade Neoplásica , Isoenzimas/metabolismo , Isoenzimas/genética
20.
Arch Pharm (Weinheim) ; 357(6): e2300718, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38466120

RESUMO

A new series of isatin-linked benzenesulfonamide derivatives (9a-w) were synthesized using the tail approach and assayed for their inhibitory potency against four different human carbonic anhydrase (hCA) isoforms, hCA I, II, IX, and XII. Most of these synthesized compounds exhibited interesting inhibition potency against isoforms hCA I, IX, and XII in the nanomolar range and by taking the standard drug acetazolamide. The most potent compounds in the case of hCA I were 9c (435.8 nM) and 9s (956.4 nM), for hCA IX, 9a (60.5 nM), 9d (95.6 nM), 9g (92.1 nM), and 9k (75.4 nM), and for hCA XII, 9p (84.5 nM). However, these compounds showed more selectivity toward hCA IX over hCA I, II, and XII. Thus, these compounds can be further developed as potential lead molecules for the development of isoform-selective hCA IX inhibitors with further structural modifications.


Assuntos
Benzenossulfonamidas , Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Desenho de Fármacos , Sulfonamidas , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Humanos , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/química , Anidrases Carbônicas/metabolismo , Estrutura Molecular , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Isatina/farmacologia , Isatina/química , Isatina/síntese química , Relação Dose-Resposta a Droga
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