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1.
Methods Mol Biol ; 2558: 221-252, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36169867

RESUMO

Proper elucidation of drug-target interaction is one of the most significant steps at the early stages of the drug development research. Computer-aided drug design tools have substantial contribution to this stage. In this chapter, we specifically concentrate on the computational methods widely used to develop reversible inhibitors for monoamine oxidase (MAO) isozymes. In this context, current computational techniques in identifying the best drug candidates showing high potency are discussed. The protocols of structure-based drug design methodologies, namely, molecular docking, in silico screening, and molecular dynamics simulations, are presented. Employing case studies of safinamide binding to MAO B, we demonstrate how to use AutoDock 4.2.6 and NAMD software packages.


Assuntos
Química Computacional , Inibidores da Monoaminoxidase , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Relação Estrutura-Atividade
2.
Int J Mol Sci ; 23(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36361903

RESUMO

Over 10 million people worldwide live with Parkinson's disease (PD) and 4% of affected people are diagnosed before the age of 50. Research on early PD-related pathways is therefore of considerable importance. Peptidylarginine deiminases (PADs) are a family of calcium-activated enzymes that, through post-translational deimination of arginine to citrulline, contribute to changes in protein function, including in pathological processes. Recent studies have highlighted roles for PADs in a range of neurological disorders including PD, but overall, investigations on PADs in Lewy body disease (LBD), including PD, are still scarce. Hence, the current pilot study aimed at performing an immunohistochemistry screen of post-mortem human brain sections from Braak stages 4-6 from PD patients, as well as patients with incidental LBD (ILBD). We assessed differences in PAD isozyme detection (assessing all five PADs), in total protein deimination/citrullination and histone H3 deimination-which is an indicator of epigenetic changes and extracellular trap formation (ETosis), which can elicit immune responses and has involvement in pathogenic conditions. The findings of our pilot study indicate that PADs and deimination are increased in cingulate cortex and hippocampus, particularly in earlier stages of the disease. PAD2 and PAD3 were the most strongly upregulated PAD isozymes, with some elevation also observed for PAD1, while PAD4 and PAD6 increase was less marked in PD brains. Total protein deimination and histone H3 deimination were furthermore increased in PD brains, with a considerable increase at earlier Braak stages, compared with controls. Our findings point to a significant contribution of PADs, which may further aid early disease biomarker discovery, in PD and other LBDs.


Assuntos
Citrulinação , Doença por Corpos de Lewy , Humanos , Desiminases de Arginina em Proteínas/metabolismo , Projetos Piloto , Histonas/metabolismo , Doença por Corpos de Lewy/metabolismo , Corpos de Lewy/metabolismo , Isoenzimas/metabolismo , Hidrolases/metabolismo
3.
Int J Mol Sci ; 23(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36362331

RESUMO

Fungal laccases play important roles in the degradation of lignocellulose. In this study, the laccase producing cotton straw medium for Pleurotus ostreatus was optimized by single-factor and orthogonal experiments, and to investigate the role of Lacc1 gene, one of the laccase-encoding genes, in the degradation of cotton straw lignin, an overexpression strain of Lacc1 gene was constructed, which was analyzed for the characteristics of lignin degradation. The results demonstrated that the culture conditions with the highest lignin degradation efficiency of the P. ostreatus were the cotton straw particle size of 0.75 mm, a solid-liquid ratio of 1:3 and containing 0.25 g/L of Tween in the medium, as well as an incubation temperature of 26 °C. Two overexpression strains (OE L1-1 and OE L1-4) of Lacc1 gene were obtained, and the gene expression increased 12.08- and 33.04-fold, respectively. The results of 1H-NMR and FTIR analyses of significant changes in lignin structure revealed that Lacc1 gene accelerated the degradation of lignin G-units and involved in the cleavage of ß-O-4 linkages and the demethylation of lignin units. These findings will help to improve the efficiency of biodelignification and expand our understanding of its mechanism.


Assuntos
Agaricales , Pleurotus , Pleurotus/genética , Pleurotus/metabolismo , Lacase/metabolismo , Lignina/metabolismo , Agaricales/metabolismo , Isoenzimas/metabolismo , Gossypium/genética , Gossypium/metabolismo
4.
Molecules ; 27(21)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36364255

RESUMO

In this work, nine new bromophenol derivatives were designed and synthesized. The alkylation reactions of (2-bromo-4,5-dimethoxyphenyl)methanol (7) with substituted benzenes 8-12 produced new diaryl methanes 13-17. Targeted bromophenol derivatives 18-21 were synthesized via the O-Me demethylation of diaryl methanes with BBr3. Moreover, the synthesized bromophenol compounds were tested with some metabolic enzymes such as acetylcholinesterase (AChE), carbonic anhydrase I (CA I), and II (CA II) isoenzymes. The novel synthesized bromophenol compounds showed Ki values that ranged from 2.53 ± 0.25 to 25.67 ± 4.58 nM against hCA I, from 1.63 ± 0.11 to 15.05 ± 1.07 nM against hCA II, and from 6.54 ± 1.03 to 24.86 ± 5.30 nM against AChE. The studied compounds in this work exhibited effective hCA isoenzyme and AChE enzyme inhibition effects. The results show that they can be used for the treatment of glaucoma, epilepsy, Parkinson's as well as Alzheimer's disease (AD) after some imperative pharmacological studies that would reveal their drug potential.


Assuntos
Acetilcolinesterase , Anidrases Carbônicas , Acetilcolinesterase/metabolismo , Anidrases Carbônicas/metabolismo , Anidrase Carbônica II , Inibidores da Anidrase Carbônica/farmacologia , Metano , Inibidores da Colinesterase/farmacologia , Isoenzimas/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular
5.
Int J Mol Sci ; 23(19)2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36233169

RESUMO

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.


Assuntos
Ouabaína , ATPase Trocadora de Sódio-Potássio , Animais , Hipóxia/metabolismo , Isoenzimas/metabolismo , Ligantes , Lipídeos , Músculo Esquelético/metabolismo , Ouabaína/metabolismo , Ouabaína/farmacologia , Ratos , ATPase Trocadora de Sódio-Potássio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
6.
Aging (Albany NY) ; 14(19): 7926-7940, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36205594

RESUMO

Acyl-CoA synthetases (ACSs) are responsible for acyl-CoA synthesis from nonpolar hydrophilic fatty acids and play a vital role in many metabolic processes. As a category of ACS isozymes, members of ACS family (AACS, ACSF2-3, AASDH) participate in lipid metabolism; however, their expression patterns, regulatory mechanisms and effects in hepatocellular carcinoma (HCC) are poorly understood. Here, through evaluating the expression profiles of ACSF gene family, we found that upregulated AACS might be more significant and valuable in development and progression of HCC. Consequently, the mRNA expression levels of AACS and ACSF2 was accordantly increased in HCC. Kaplan-Meier plotter revealed that HCC patients with high level of AACS were highly related to a shorter overall survival time and relapse-free survival. Genetic alterations using cBioPortal revealed that the alteration rate of AACS were 5%. We also found that the functions of ACSF gene family were linked to several cancer-associated pathways, including long-term potentiation, phospholipase D signaling pathway and purine metabolism. TIMER database indicated that the AACS and ACSF2 had a strong relationship with the infiltration of six types of immune cells (macrophages, neutrophils, CD8+ T-cells, B-cells, CD4+ T-cells and dendritic cells). Next, Diseasemeth database revealed that the global methylation levels of ACSF2 was higher in HCC patients. In conclusion, this study firstly demonstrated that Acyl-CoA synthesis gene family, in particular, AACS, could be associated with immune microenvironment, thereby influencing the development and prognosis of patients with HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Fosfolipase D , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Isoenzimas/genética , Isoenzimas/metabolismo , Recidiva Local de Neoplasia , Prognóstico , Coenzima A Ligases/genética , Biomarcadores , RNA Mensageiro/metabolismo , Ácidos Graxos , Purinas , Coenzima A , Biomarcadores Tumorais/genética , Microambiente Tumoral/genética
7.
Genes (Basel) ; 13(10)2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36292720

RESUMO

Lactate dehydrogenase (LDH) catalyzes the reversible conversion of L-lactate to pyruvate. LDH-A deficiency is an autosomal recessive disorder (glycogenosis type XI, OMIM#612933) caused by mutations in the LDHA gene. We present two young adult female patients presenting with intolerance to anaerobic exercise, episodes of rhabdomyolysis, and, in one of the patients, psoriasis-like dermatitis. We identified in the LDHA gene a homozygous c.410C>A substitution that predicts a p.Ser137Ter nonsense mutation in Patient One and a compound heterozygous c.410C>A (p.Ser137Ter) and c.750G>A (p.Trp250Ter) nonsense mutation in Patient Two. The pathogenicity of the variants was demonstrated by electrophoretic separation of LDH isoenzymes. Moreover, a flat lactate curve on the forearm exercise test, along with the clinical combination of myopathy and psoriatic-like dermatitis, can also lead to the diagnosis.


Assuntos
Dermatite , Doença de Depósito de Glicogênio , Humanos , Feminino , Lactato Desidrogenase 5 , Isoenzimas/genética , Isoenzimas/metabolismo , Códon sem Sentido , Ácido Láctico/metabolismo , Ácido Pirúvico , Mutação
8.
Molecules ; 27(19)2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36235158

RESUMO

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography-tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.


Assuntos
Solanum tuberosum , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Catalase/metabolismo , Frutoquinases/análise , Frutoquinases/metabolismo , Isoenzimas/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos/química , Proteoma/metabolismo , Proteômica/métodos , Solanum tuberosum/química , Espectrometria de Massas em Tandem
9.
Biochemistry ; 61(20): 2229-2240, 2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36197914

RESUMO

α-Carboxyketose synthases, including 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase , Isoenzimas , 3-Desoxi-7-Fosfo-Heptulonato Sintase/química , Sítios de Ligação , Catálise , Comunicação , Cristalografia por Raios X , Deutério , Escherichia coli , Humanos , Isoenzimas/metabolismo , Ligantes , Fenilalanina/metabolismo , Fosfatos
10.
Semin Cancer Biol ; 86(Pt 2): 93-100, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36096316

RESUMO

The energy metabolism of tumor cells is considered one of the hallmarks of cancer because it is different from normal cells and mainly consists of aerobic glycolysis, fatty acid oxidation, and glutaminolysis. It is about one hundred years ago since Warburg observed that cancer cells prefer aerobic glycolysis even in normoxic conditions, favoring their high proliferation rate. A pivotal enzyme driving this phenomenon is lactate dehydrogenase (LDH), and this review describes prognostic and therapeutic opportunities associated with this enzyme, focussing on tumors with limited therapeutic strategies and life expectancy (i.e., pancreatic and thoracic cancers). Expression levels of LDH-A in pancreatic cancer tissues correlate with clinicopathological features: LDH-A is overexpressed during pancreatic carcinogenesis and showed significantly higher expression in more aggressive tumors. Similarly, LDH levels are a marker of negative prognosis in patients with both adenocarcinoma or squamous cell lung carcinoma, as well as in malignant pleural mesothelioma. Additionally, serum LDH levels may play a key role in the clinical management of these diseases because they are associated with tissue damage induced by tumor burden. Lastly, we discuss the promising results of strategies targeting LDH as a treatment strategy, reporting recent preclinical and translational studies supporting the use of LDH-inhibitors in combinations with current/novel chemotherapeutics that can synergistically target the oxygenated cells present in the tumor.


Assuntos
Metabolismo Energético , Lactato Desidrogenase 5 , Neoplasias Pancreáticas , Neoplasias Torácicas , Humanos , Glicólise/fisiologia , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Neoplasias Torácicas/metabolismo
11.
Chemosphere ; 308(Pt 2): 136349, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36084836

RESUMO

Chiral polychlorinated biphenyls (PCBs) have atropisomers that have different axial chiralities and exist as racemic mixtures. However, biochemical processes often result in the unequal accumulation of these atropisomers in organisms. This phenomenon leads to enantiospecific toxicity enhancement or reduction because either of the atropisomers mainly affects toxicity expression. Enantioselective accumulation is caused by cytochrome P450 (CYP, P450) monooxygenases, especially the CYP2B subfamilies. Therefore, this study investigates the metabolism of a chiral PCB in vitro. Both atropisomers isolated from racemic 2,2',3,4,4',5',6-heptachlorobiphenyl (CB183) were metabolized by human CYP2B6, but not rat CYP2B1. This may be due to the difference in the size of the substrate-binding cavities of CYP2B6 and CYP2B1. The stable accommodation of (-)-CB183 in the cavity without any steric hindrance explained the preferential metabolism of (-)-CB183 compared to (+)-CB183. Two hydroxylated metabolites, 3'-OH-CB183 and 5-OH-CB183, were identified. The docking study showed that the 3'-position of the trichlorophenyl ring closely approaches the heme of CYP2B6. To our knowledge, this is the first study to elucidate the structural basis of chiral PCB metabolism by P450 isozymes. These results will help promote the precise toxicity evaluation of chiral PCBs and provide an explanation of the structural basis of chiral PCB metabolism.


Assuntos
Bifenilos Policlorados , Animais , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme , Humanos , Hidroxilação , Isoenzimas/metabolismo , Bifenilos Policlorados/química , Ratos , Estereoisomerismo
12.
Bioorg Med Chem ; 73: 116988, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36113282

RESUMO

A simplified analog (3) of aplysiatoxin was synthesized. Compound 3 has only one tetrahydropyran ring at positions 3-7, the A-ring of the spiroketal moiety, which is the conformation-controlling unit for the macrolactone ring. Nuclear magnetic resonance (NMR) analysis and density functional theory (DFT) calculations indicated that 3 existed as an equilibrium mixture of two conformers arising from inversion of the chair conformation of the 2,6-trans-tetrahydropyran ring. The des-B-ring analog 3 binds protein kinase C isozymes and exhibits antiproliferative activity toward human cancer cell lines, comparable to 18-deoxy-aplog-1 with a spiroketal moiety.


Assuntos
Antineoplásicos , Isoenzimas , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Furanos , Humanos , Isoenzimas/metabolismo , Toxinas de Lyngbya , Proteína Quinase C/metabolismo , Compostos de Espiro , Relação Estrutura-Atividade
13.
Glycobiology ; 32(12): 1153-1163, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36106687

RESUMO

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a ß-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind ß-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with ß-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a ß-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with ß-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of ß-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.


Assuntos
Acetilglucosamina , Isoenzimas , Animais , Humanos , Acetilglucosamina/metabolismo , Isoenzimas/metabolismo , Filogenia , N-Acetilglucosaminiltransferases/genética , Glicosiltransferases/metabolismo , Polissacarídeos/química , Manose/química
14.
Biochem Pharmacol ; 204: 115243, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36084709

RESUMO

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.


Assuntos
Exantema , Infecções por HIV , Sulfotransferases/metabolismo , Animais , Arilsulfotransferase/genética , Arilsulfotransferase/metabolismo , Citosol/metabolismo , Exantema/metabolismo , Infecções por HIV/metabolismo , Humanos , Isoenzimas/metabolismo , Nevirapina/metabolismo , Polimorfismo Genético , Ratos , Sulfatos/metabolismo , Sulfotransferases/genética
15.
Int J Mol Sci ; 23(18)2022 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-36142836

RESUMO

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.


Assuntos
Ouabaína , ATPase Trocadora de Sódio-Potássio , Animais , Corticosterona/metabolismo , Diafragma/metabolismo , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Ligantes , Masculino , Músculo Esquelético/metabolismo , Ouabaína/metabolismo , Ouabaína/farmacologia , Ratos , Ratos Wistar , Solução Salina , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
16.
Plant Physiol Biochem ; 190: 70-80, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36099810

RESUMO

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.


Assuntos
Arabidopsis , Fosfoenolpiruvato Carboxilase , Trifosfato de Adenosina , Ácidos Carboxílicos , Isoenzimas/genética , Isoenzimas/metabolismo , Lipídeos , Nitratos , Nitrogênio/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas Serina-Treonina Quinases , Sementes
17.
J Biol Chem ; 298(10): 102466, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36087841

RESUMO

The internalization of G protein-coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.


Assuntos
Isoenzimas , Proteína Quinase C , Animais , Humanos , Ratos , Sistemas CRISPR-Cas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Isoenzimas/genética , Isoenzimas/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Técnicas de Inativação de Genes
18.
Proc Natl Acad Sci U S A ; 119(39): e2204396119, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36122218

RESUMO

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.


Assuntos
Fosfatidilserinas , Esfingosina , Ceramidas/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Isoenzimas/metabolismo , Lipossomos/metabolismo , Lisofosfolipídeos , Fosfatidilserinas/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
19.
Biochem Biophys Res Commun ; 630: 30-35, 2022 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-36130444

RESUMO

Casein kinase 2 (CK2) is a vital protein kinase that consists of two catalytic subunits (CK2α1 and/or CK2α2) and two regulatory subunits (CK2ß). CK2α1 is a drug target for nephritis and cancers, while CK2α2 is a serious off-target because its inhibition causes testicular toxicity. High similarity between the isozymes CK2α1 and CK2α2 make it difficult to design CK2α1-specific inhibitors. Herein, the crystal structures of CK2α1 and CK2α2 complexed with a 3-amino-pyrazole inhibitor revealed the remarkable differences in the protein-inhibitor interaction modes. This inhibitor bound to the ATP binding sites of both isozymes in apparently distinct orientations. In addition, another molecule of this inhibitor bound to CK2α1, but not to CK2α2, at the CK2ß protein-protein interface. Binding energy calculations and biochemical experiments suggested that this inhibitor possesses the conventional ATP-competitive characteristics with moderate allosteric function in a molecular glue mechanism. These results will assist the potential design of potent and selective CK2α1 inhibitors.


Assuntos
Caseína Quinase II , Isoenzimas , Pirazóis/farmacologia , Trifosfato de Adenosina , Caseína Quinase II/metabolismo , Isoenzimas/metabolismo , Inibidores de Proteínas Quinases/farmacologia
20.
Biopharm Drug Dispos ; 43(5): 213-217, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36151066

RESUMO

The estimation of the contributions of UDP-glucuronosyl transferase (UGT) isoforms to the overall metabolism still suffers from technical difficulties due to limited information on enzyme levels in recombinant systems and specific inhibitors, unlike the case for cytochrome P450s (CYPs). The protein expression levels of UGT in both recombinant system microsomes (RM) and human liver microsomes (HLM) were quantified using liquid chromatography-tandem mass spectrometry, and the relative expression factor (REF) value of HLM to recombinant microsomes was estimated to evaluate the fractions of drug metabolism by a single UGT enzyme (fmUGT) of UGT substrates. The REF values of UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17 were 0.228, 0.0714, 0.0665, 0.420, 0.118, and 0.0442, respectively. fmUGTs in HLM were estimated for several typical UGT substrates utilizing these values and metabolic clearances in RM. These values were comparable to the reported values estimated by various methods. This study provided useful information on REF values, which promote a robust estimation of fmUGT values for UGT substrates when evaluating the contribution of UGT isoforms to total metabolic clearance.


Assuntos
Glucuronosiltransferase , Isoenzimas , Humanos , Isoenzimas/metabolismo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/metabolismo , Taxa de Depuração Metabólica , Cromatografia Líquida , Difosfato de Uridina/metabolismo , Glucuronídeos/metabolismo
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