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1.
Plant Sci ; 312: 111033, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34620437

RESUMO

The glutamine amidotransferase gene GAT1_2.1 is a marker of N status in Arabidopsis root, linked to a shoot branching phenotype. The protein has an N-terminal glutamine amidotransferase domain and a C-terminal extension with no recognizable protein domain. A purified, recombinant version of the glutamine amidotransferase domain was catalytically active as a glutaminase, with apparent Km value of 0.66 mM and Vmax value of 2.6 µkatal per mg. This form complemented an E. coli glutaminase mutant, ΔYneH. Spiking of root metabolite extracts with either the N-terminal or full length form purified from transformed tobacco leaves led to reciprocal changes in glutamine and ammonia concentration. No product derived from amido-15N-labeled glutamine was identified. Visualization of GAT1_2.1-YPF transiently expressed in tobacco leaves confirmed its mitochondrial localization. gat1_2.1 exhibited reduced growth as compared with wild-type seedlings on media with glutamine as sole nitrogen source. Results of targeted metabolite profiling pointed to a possible activation of the GABA shunt in the mutant following glutamine treatments, with reduced levels of glutamic acid, 2-oxoglutarate and γ-aminobutyric acid and increased levels of succinic acid. GAT1_2.1 may act as a glutaminase, in concert with Glutamate Dehydrogenase 2, to hydrolyze glutamine and channel 2-oxoglutarate to the TCA cycle under high nitrogen conditions.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Glutaminase/genética , Glutaminase/metabolismo , Nitrogênio/metabolismo , Raízes de Plantas/enzimologia , Transaminases/genética , Transaminases/metabolismo , Variação Genética , Genótipo , Raízes de Plantas/genética
2.
J Chem Inf Model ; 61(11): 5569-5580, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34653331

RESUMO

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.


Assuntos
Simulação de Dinâmica Molecular , Transaminases , Simulação de Acoplamento Molecular , Especificidade por Substrato , Transaminases/metabolismo , Vibrio
3.
Nutr Metab Cardiovasc Dis ; 31(11): 3210-3218, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34511290

RESUMO

BACKGROUND AND AIM: Circulating amino acids are modified by sex, body mass index (BMI) and insulin resistance (IR). However, whether the presence of genetic variants in branched-chain amino acid (BCAA) catabolic enzymes modifies circulating amino acids is still unknown. Thus, we determined the frequency of two genetic variants, one in the branched-chain aminotransferase 2 (BCAT2) gene (rs11548193), and one in the branched-chain ketoacid dehydrogenase (BCKDH) gene (rs45500792), and elucidated their impact on circulating amino acid levels together with clinical, anthropometric and biochemical parameters. METHODS AND RESULTS: We performed a cross-sectional comparative study in which we recruited 1612 young adults (749 women and 863 men) aged 19.7 ± 2.1 years and with a BMI of 24.9 ± 4.7 kg/m2. Participants underwent clinical evaluation and provided blood samples for DNA extraction and biochemical analysis. The single nucleotide polymorphisms (SNPs) were determined by allelic discrimination using real-time polymerase chain reaction (PCR). The frequencies of the less common alleles were 15.2 % for BCAT2 and 9.83 % for BCKDH. The subjects with either the BCAT2 or BCKDH SNPs displayed no differences in the evaluated parameters compared with subjects homozygotes for the most common allele at each SNP. However, subjects with both SNPs had higher body weight, BMI, blood pressure, glucose, and circulating levels of aspartate, isoleucine, methionine, and proline than the subjects homozygotes for the most common allele (P < 0.05, One-way ANOVA). CONCLUSION: Our findings suggest that the joint presence of both the BCAT2 rs11548193 and BCKDH rs45500792 SNPs induces metabolic alterations that are not observed in subjects without either SNP.


Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Aminoácidos/sangue , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo de Nucleotídeo Único , Proteínas da Gravidez/genética , Transaminases/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Adolescente , Fatores Etários , Biomarcadores/sangue , Glicemia/análise , Pressão Sanguínea , Índice de Massa Corporal , Estudos Transversais , Feminino , Frequência do Gene , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , México , Antígenos de Histocompatibilidade Menor/metabolismo , Fenótipo , Proteínas da Gravidez/metabolismo , Transaminases/metabolismo , Adulto Jovem
4.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544857

RESUMO

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2 Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking up-regulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed up-regulation of IL-6 transcription and secretion compared to wild-type cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wild-type cells. Treatment with anti-IL-6 (αIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2 +/- mice while reducing PSAT1 expression. These data reveal a mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.


Assuntos
Interleucina-6/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina/metabolismo , Transaminases/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Animais , Interleucina-6/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transaminases/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/fisiologia
5.
Life Sci ; 285: 119986, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34592233

RESUMO

AIMS: Hepatic ischemia reperfusion injury (HIRI) is a complication of liver surgery and liver transplantation. Adipose-derived stem cells (ADSCs) can inhibit oxidative stress and inflammation through a paracrine effect. This study aimed to determine the optimal time window of ADSCs transplantation to restore liver function after HIRI. MAIN METHODS: A rat model of hepatic ischemia reperfusion combined with partial hepatectomy (HIR/PH) was established. The animals were injected intravenously with 2 × 106 rat ADSCs 2 h before, immediately after, or 6 h after surgery. Liver tissues and blood samples were collected for routine histological and biochemical assays. The molecular changes were analyzed by qRT-PCR and western blotting. KEY FINDINGS: ADSCs significantly improved liver tissue structure and decreased the levels of AST, ALT and ALP, which was indicative of functional recovery. In addition, transplantation of ADSCs immediately after operation decreased the levels of inflammation-related cytokines such as TNF-α, IL-1ß and IL-6, and significantly increased the activity of antioxidant enzymes. At the same time, the expression of MDA was decreased. Mechanistically, ADSCs activated the Keap1/Nrf2 pathway in the injured liver. Transplantation of ADSCs pre- and 6 h post-operation did not significantly affect some indices such as mRNA and protein expression of HO-1, and protein expression of NQO1. SIGNIFICANCE: Transplanting ADSCs immediately after surgery accelerated tissue repair and functional recovery of the liver by activating the Keap1/Nrf2 pathway, which inhibited hepatic inflammation and oxidative stress, and restored the hepatic microenvironment.


Assuntos
Hepatectomia/efeitos adversos , Regeneração Hepática , Transplante de Fígado/efeitos adversos , Fígado/irrigação sanguínea , Fígado/cirurgia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/cirurgia , Tecido Adiposo/citologia , Alanina Transaminase/metabolismo , Animais , Modelos Animais de Doenças , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/enzimologia , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Fosfodiesterase I/metabolismo , Ratos , Ratos Sprague-Dawley , Transaminases/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445250

RESUMO

The combined impact of an increasing demand for liver transplantation and a growing incidence of nonalcoholic liver disease has provided the impetus for the development of innovative strategies to preserve steatotic livers. A natural oxygen carrier, HEMO2life®, which contains M101 that is extracted from a marine invertebrate, has been used for static cold storage (SCS) and has shown superior results in organ preservation. A total of 36 livers were procured from obese Zucker rats and randomly divided into three groups, i.e., control, SCS-24H and SCS-24H + M101 (M101 at 1 g/L), mimicking the gold standard of organ preservation. Ex situ machine perfusion for 2 h was used to evaluate the quality of the livers. Perfusates were sampled for functional assessment, biochemical analysis and subsequent biopsies were performed for assessment of ischemia-reperfusion markers. Transaminases, GDH and lactate levels at the end of reperfusion were significantly lower in the group preserved with M101 (p < 0.05). Protection from reactive oxygen species (low MDA and higher production of NO2-NO3) and less inflammation (HMGB1) were also observed in this group (p < 0.05). Bcl-1 and caspase-3 were higher in the SCS-24H group (p < 0.05) and presented more histological damage than those preserved with HEMO2life®. These data demonstrate, for the first time, that the addition of HEMO2life® to the preservation solution significantly protects steatotic livers during SCS by decreasing reperfusion injury and improving graft function.


Assuntos
Fígado Gorduroso/metabolismo , Hemoglobinas/farmacologia , Fígado/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Caspase 3/metabolismo , Ciclina D1/metabolismo , Fígado Gorduroso/patologia , Proteína HMGB1/metabolismo , Ácido Láctico/metabolismo , Masculino , Ratos , Ratos Zucker , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transaminases/metabolismo
7.
Molecules ; 26(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34443642

RESUMO

Among industrially important pyridoxal-5'-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.


Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Bacteroidetes/metabolismo , Domínio Catalítico/fisiologia , Transaminases/metabolismo , Cristalografia por Raios X/métodos , Modelos Moleculares , Fosfato de Piridoxal/metabolismo , Especificidade por Substrato/fisiologia
8.
Biomed Res Int ; 2021: 2593748, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34447850

RESUMO

Artificial intelligence technologies such as machine learning have been applied to protein engineering, with unique advantages in protein structure, function prediction, catalytic activity, and other issues in recent years. Screening better mutants is still a bottleneck in protein engineering. In this paper, a new sequence-activity relationship method was analyzed for its application in improving the thermal stability of Aspergillus terreus (R)-ω-selective amine transaminase. The experimental data from 6 single-point mutated enzymes were used as a learning dataset to build models and predict the thermostability of 26 mutants. Based on digital signal processing (DSP), this method digitized the amino acid sequence of proteins by fast Fourier transform (FFT) and then established the best model applying partial least squares regression (PLSR) to screen out all possible mutants, especially those with high performance. In protein engineering, the innovative sequence activity relationship (ISAR) method can make a reasonable prediction using limited experimental data and significantly reduce the experimental cost. The half-life (T 1/2) of (R)-ω-transaminase was fitted with the amino acid sequence by the ISAR algorithm, resulting in an R 2 of 0.8929 and a cvRMSE of 4.89. At the same time, the mutants with higher T 1/2 than the existing ones were predicted, laying the groundwork for better (R)-ω-transaminase in the later stage. The ISAR algorithm is expected to provide a new technique for protein evolution and screening.


Assuntos
Aminas/química , Aspergillus/enzimologia , Aprendizado de Máquina , Transaminases/química , Aminas/metabolismo , Aspergillus/química , Temperatura Alta , Modelos Moleculares , Engenharia de Proteínas/métodos , Estabilidade Proteica , Elementos Estruturais de Proteínas , Especificidade por Substrato , Transaminases/metabolismo
9.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202732

RESUMO

The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Botrytis , Resistência à Doença , Interações Hospedeiro-Patógeno , Metabolômica , Doenças das Plantas/microbiologia , Trichoderma , Biologia Computacional/métodos , Ácido Glutâmico/metabolismo , Metabolômica/métodos , Fenótipo , Desenvolvimento Vegetal , Simbiose , Transaminases/genética , Transaminases/metabolismo
10.
Biochim Biophys Acta Mol Cell Res ; 1868(11): 119114, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34329662

RESUMO

IDH1 mutations are frequent and early events in gliomas. Mutant IDH1 produces D-2HG that causes epigenetic changes by increasing histone and DNA methylations, thereby contributing to tumor growth. Mutant IDH1 rewires metabolism and endows a few therapeutic vulnerabilities in cells. But, mutant IDH1 inhibitor(s) treatments reverse these therapeutic vulnerabilities by increasing cell growth. Nevertheless, it is unclear how mutant IDH1 inhibitor(s) increases cell growth. As mutant IDH1 inhibitor(s) increase cell growth, therefore we asked whether mutant IDH1 inhibitor(s) activate oncogenes in mutant IDH1-expressing cells. To answer this question, we used allosteric mutant IDH1 inhibitors to treat mutant IDH1-expressing HT1080 cells, and examined for activation of oncogenes by assessing the levels of our read-outs: BCAT1 and YKL-40. We found that mutant IDH1 inhibitors' treatments increased BCAT1 and YKL-40 levels in HT1080 cells. Next, we observed that mutant IDH1 inhibitors activated STAT3 by phosphorylation at Tyr-705 position (pSTAT3-Y705) and its nuclear translocation. Upon examining the molecular mechanism of pSTAT3-Y705 activation in mutant IDH1 inhibitor-treated cells, we found that mutant IDH1 strongly bound STAT3, but mutant IDH1 inhibitor treatment decreased mutant IDH1-STAT3 binding. Furthermore, we observed that STAT3-knockdown and pharmacological inhibition of STAT3 attenuated the mutant IDH1 inhibitor-mediated increase in BCAT1 and YKL-40 levels, whereas STAT3 overexpression and Interleukin-6 (STAT3 activator) treatments increased BCAT1 and YKL-40 levels. We conclude that mutant IDH1 inhibitors activate the oncogenic transcription factor-STAT3 leading to an increase in BCAT1 and YKL-40 levels in mutant IDH1-expressing cells.


Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transaminases/metabolismo , Tirosina/metabolismo , Benzenoacetamidas/farmacologia , Células Cultivadas , Humanos , Imidazóis/farmacologia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Fosforilação
11.
mBio ; 12(3): e0076821, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34154419

RESUMO

Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding ß-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both ß-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB. IMPORTANCE Branched-chain amino acid (BCAA) biosynthesis is important for pathogenic fungi to successfully cause disease in human and plant hosts. The enzymes for their production are absent from humans and, therefore, provide potential antifungal targets. While BCAA biosynthesis is well characterized in yeasts, it is poorly understood in filamentous fungal pathogens. Developing a thorough understanding of both the genes encoding the metabolic enzymes for BCAA biosynthesis and how their expression is regulated will inform target selection for antifungal drug development.


Assuntos
Aminoácidos de Cadeia Ramificada/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Aspergillus nidulans/genética , Vias Biossintéticas/genética , Aminoácidos de Cadeia Ramificada/biossíntese , Aspergillus nidulans/química , Regulação Fúngica da Expressão Gênica , Leucina/biossíntese , Transaminases/genética , Transaminases/metabolismo
12.
Biochemistry ; 60(24): 1926-1932, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096710

RESUMO

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.


Assuntos
Organofosfonatos/química , Fosfitos/química , Bacillus subtilis/metabolismo , Catálise , Glucosamina/biossíntese , Glucosamina/química , Glucosamina/metabolismo , Glucose/metabolismo , Glucose-6-Fosfato , Cinética , Organofosfonatos/metabolismo , Oxirredução , Fosfitos/metabolismo , Transaminases/metabolismo , Xilose/metabolismo
13.
Mol Med Rep ; 24(2)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34109436

RESUMO

Cytarabine is a key chemotherapy drug for treating leukemia; however, chemotherapy­induced multidrug resistance is a major cause of therapy failure or tumor recurrence. Current medical treatment strategies still cannot address the issue of multidrug resistance phenotypes in the treatment of leukemia. Curcumin counteracts tumor development by inducing apoptosis in cytarabine­resistant acute myeloid leukemia cells. Branched­chain amino acid transaminase 1 (BCAT1), an aminotransferase enzyme, acts on branched­chain amino acids. Moreover, the aberrant expression of BCAT1 has been observed in numerous cancer cells, and BCAT1 serves a critical role in the progression of myeloid leukemia. BCAT1 can interfere with cancer cell proliferation by regulating mTOR­mediated mitochondrial biogenesis and function. The present study aimed to investigate whether curcumin induces apoptosis by regulating BCAT1 expression and mTOR signaling in cytarabine­resistant myeloid leukemia cells. Four leukemia cell lines and three primary myeloid leukemia cells were treated with curcumin, and the expression and activity of BCAT1 and mTOR were investigated by reverse transcription­quantitative PCR, western blotting and α­KG quantification assay. The results demonstrated that curcumin inhibited BCAT1 expression in Kasumi­1, KG­1, HL60, cytarabine­resistant HL60, and cytarabine­resistant primary myeloid leukemia cells. Notably, tetrahydrocurcumin, a major metabolite of curcumin, and cytarabine had no inhibitory effect on BCAT1 expression. Furthermore, BCAT1 and mTOR signaling may modulate each other in cytarabine­resistant HL60 cells. The present results indicated that curcumin may induce apoptosis by inhibiting the BCAT1 and mTOR pathways. Thus, understanding the mechanism underlying curcumin­induced apoptosis in cytarabine­resistant cells can support the development of novel drugs for leukemia.


Assuntos
Curcumina/farmacologia , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transaminases/antagonistas & inibidores , Adolescente , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Indóis/farmacologia , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Purinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Transaminases/genética , Transaminases/metabolismo
14.
PLoS One ; 16(6): e0250368, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061870

RESUMO

BACKGROUND: Isoniazid preventive therapy (IPT) reduces tuberculosis reactivation and mortality among persons living with HIV (PLWH), yet hepatotoxicity concerns exclude "regular and heavy alcohol drinkers" from IPT. We aimed to determine the prevalence of elevated liver transaminases among PLWH on antiretroviral therapy (ART) who engage in alcohol use. SETTING: The Immune Suppression Syndrome Clinic of Mbarara, Uganda. METHODS: We defined elevated liver transaminases as ≥1.25 times (X) the upper limit of normal (ULN) for alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST). We evaluated the associations of current alcohol use and other variables of interest (sex, body mass index, and ART regimen) with elevated transaminases at study screening, using multivariable logistic regression to obtain adjusted odds ratios (aOR) and 95% confidence intervals (CI). RESULTS: Among 1301 participants (53% female, median age 39 years, 67.4% current alcohol use), 18.8% (95% CI: 16.8-21.1) had elevated transaminases pre-IPT, with few (1.1%) severe (≥5X the ULN). The proportion with any elevation among those currently using alcohol and those abstaining was 22.3% and 11.6%, respectively (p<0.01). In multivariable analyses, those currently using alcohol had higher odds of elevated transaminases compared to those abstaining (aOR 1.65, 95% CI 1.15-2.37) as did males compared to females (aOR 2.68, 95% CI 1.90-3.78). CONCLUSIONS: Pre-IPT elevated transaminases among PLWH receiving ART were common, similar to prior estimates, but severe elevations were rare. Current drinking and male sex were independently associated with elevated transaminases. Further research is needed to determine the implications of such transaminase elevations and alcohol use on providing IPT.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Fígado/enzimologia , Transaminases/metabolismo , Adolescente , Adulto , Fármacos Anti-HIV/uso terapêutico , Feminino , Infecções por HIV/complicações , Humanos , Fígado/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Prevalência , Uganda
15.
Clin Nutr ; 40(5): 2609-2619, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33933727

RESUMO

BACKGROUND & AIMS: Regular consumption of fast-food (FF) as a form of typical Western style diet is associated with obesity and the metabolic syndrome, including its hepatic manifestation nonalcoholic fatty liver disease. Currently, it remains unclear how intermittent excess FF consumption may influence liver metabolism. The study aimed to characterize the effects of a single FF binge on hepatic steatosis, inflammation, bile acid (BA), glucose and lipid metabolism. METHODS: Twenty-five healthy individuals received a FF meal and were asked to continue eating either for a two-hour period or until fully saturated. Serum levels of transaminases, fasting BA, lipid profile, glucose and cytokine levels as well as transient elastography and controlled attenuation parameter (CAP; to assess hepatic steatosis) were analyzed before (day 0) and the day after FF binge (day 1). Feces was collected prior and after the FF challenge for microbiota analysis. RESULTS: The FF meal induced a modest increase in CAP, which was accompanied by a robust increase of fasting serum BA levels. Surprisingly, levels of cholesterol and bilirubin were significantly lower after the FF meal. Differentiating individuals with a relevant delta BA (>1 µmol/l) increase vs. individuals without (delta BA ≤1 µmol/l), identified several gut microbiota, as well as gender to be associated with the BA increase and the observed alterations in liver function, metabolism and inflammation. CONCLUSION: A single binge FF meal leads to a robust increase in serum BA levels and alterations in parameters of liver injury and metabolism, indicating a novel metabolic aspect of the gut-liver axis.


Assuntos
Ácidos e Sais Biliares/química , Metabolismo Energético , Fast Foods , Microbioma Gastrointestinal , Inflamação/etiologia , Adulto , Bilirrubina , Fezes/microbiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Fatores Sexuais , Transaminases/metabolismo , Adulto Jovem
16.
J Med Chem ; 64(10): 6730-6744, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33955740

RESUMO

Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3-6. Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29, a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31, which has weaker potency and increased permeability.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , DNA/química , Bibliotecas de Moléculas Pequenas/química , Oxirredutases do Álcool/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , DNA/metabolismo , Desenho de Fármacos , Meia-Vida , Humanos , Hiperoxalúria Primária/metabolismo , Hiperoxalúria Primária/patologia , Indóis/química , Indóis/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/metabolismo , Transaminases/genética , Transaminases/metabolismo
17.
Biochemistry ; 60(20): 1609-1618, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33949189

RESUMO

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.


Assuntos
Amônia-Liases/metabolismo , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Fosfato de Piridoxal/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Glucosamina/metabolismo , Glucose-6-Fosfato/metabolismo , Cinética , Liases/metabolismo , Modelos Moleculares , Fosfatos , Bases de Schiff , Especificidade por Substrato , Transaminases/metabolismo
18.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924837

RESUMO

It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the "closed" state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase's function and new modality to modulate enzymatic efficiency.


Assuntos
Glicosiltransferases/metabolismo , Transaminases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Transaminases/química , Transaminases/genética
19.
J Biol Chem ; 296: 100643, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33862086

RESUMO

Coenzyme Q (CoQ), a redox-active lipid essential for oxidative phosphorylation, is synthesized by virtually all cells, but how eukaryotes make the universal CoQ head group precursor 4-hydroxybenzoate (4-HB) from tyrosine is unknown. The first and last steps of this pathway have been defined in Saccharomyces cerevisiae, but the intermediates and enzymes involved in converting 4-hydroxyphenylpyruvate (4-HPP) to 4-hydroxybenzaldehyde (4-HBz) have not been described. Here, we interrogate this pathway with genetic screens, targeted LC-MS, and chemical genetics. We identify three redundant aminotransferases (Bna3, Bat2, and Aat2) that support CoQ biosynthesis in the absence of the established pathway tyrosine aminotransferases, Aro8 and Aro9. We use isotope labeling to identify bona fide tyrosine catabolites, including 4-hydroxyphenylacetate (4-HPA) and 4-hydroxyphenyllactate (4-HPL). Additionally, we find multiple compounds that rescue this pathway when exogenously supplemented, most notably 4-hydroxyphenylacetaldehyde (4-HPAA) and 4-hydroxymandelate (4-HMA). Finally, we show that the Ehrlich pathway decarboxylase Aro10 is dispensable for 4-HB production. These results define new features of 4-HB synthesis in yeast, demonstrate the redundant nature of this pathway, and provide a foundation for further study.


Assuntos
Parabenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Tirosina/metabolismo , Ubiquinona/análogos & derivados , Oxirredução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transaminases/genética , Ubiquinona/metabolismo
20.
Life Sci ; 276: 119405, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33798550

RESUMO

AIMS: Gastric cancer stem cells (GCSCs) have been used as a therapeutic target. This study aims to estimate the role of miR-98-5p (termed miR-98) in the development of GCSCs. MAIN METHODS: The expression of miR-98 in CD44+ GCSCs was verified by RT-PCR. The miR-98 was overexpressed in CD44+ GCSCs by Lentivirus. The ability of self-renewal, invasion, chemoresistance and tumorigenicity was detected in vitro or in vivo after overexpression of miR-98. The target genes of miR-98 were predicted and verified by luciferase reporter assays. The effects miR-98/BCAT1 signaling on the chemoresistance and tumorigenicity of CD44+ GCSCs were investigated in a xenograft model by rescue experiments. KEY FINDINGS: We have shown that miR-98 was decreased in CD44+ GCSCs. The overexpression of miR-98 could inhibit the expression of stem-related genes and the ability of self-renewal, invasion, and tumorigenicity of GCSCs. Also, we found that miR-98 overexpression enhances the sensitivity to cisplatin treatment in vitro. Using a xenograft model, we showed that miR-98 overexpression reversed paclitaxel resistance to CD44+ GCSCs. Finally, we found that branched-chain aminotransferases 1 (BCAT1) is a target gene of miR-98. Overexpressed BCAT1 reversed xenograft tumor formation ability and attenuated the paclitaxel chemosensitivity induced by miR-98 downregulation. Furthermore, BCAT1 restoration affected the expression of invasion and drug resistance-related genes. SIGNIFICANCE: This study revealed miR-98 inhibits gastric cancer cell stemness and chemoresistance by targeting BCAT1, suggesting that this miR-98/BCAT1 axis represents a potential therapeutic target in gastric cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Transaminases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Cisplatino/farmacologia , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transaminases/genética , Transaminases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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