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1.
PLoS One ; 15(10): e0232645, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33108364

RESUMO

Boosting energy expenditure by harnessing the activity of brown adipocytes is a promising strategy for combatting the global epidemic of obesity. Many studies have revealed that the ß3-adrenergic receptor agonist is a potent activator of brown adipocytes, even in humans, and PKA and p38 MAPK have been demonstrated for regulating the transcription of a wide range of critical genes such as Ucp1. We previously revealed that the PKA-ASK1-p38 axis is activated in immature brown adipocytes and contributes to functional maturation. However, the downstream mechanisms of PKA that initiate the p38 MAPK cascade are still mostly unknown in mature brown adipocytes. Here, we identified the ASK family as a crucial signaling molecule bridging PKA and MAPK in mature brown adipocytes. Mechanistically, the phosphorylation of ASK1 at threonine 99 and serine 993 is critical in PKA-dependent ASK1 activation. Additionally, PKA also activates ASK2, which contributes to MAPK regulation. These lines of evidence provide new details for tailoring a ßAR-dependent brown adipocyte activation strategy.


Assuntos
Adipócitos Marrons/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Metabolismo Energético , Ativação Enzimática , Regulação da Expressão Gênica , Células HEK293 , Humanos , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinases/genética , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Fosforilação , Serina/metabolismo , Treonina/metabolismo
2.
Poult Sci ; 99(5): 2508-2518, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359587

RESUMO

The present study was conducted to investigate the effects of genetic selection and threonine levels on meat quality in Pekin ducks. At 15 D of age, 192 lean ducks and 192 fatty ducks were selected and allotted to one of three treatments with 8 replicates with similar BW (8 ducks/cage), respectively. All ducks were fed the experimental diets (0.00, 0.15, and 0.30% added threonine) for 21 D from 15 to 35 D of age. The results showed that fatty ducks had higher (P < 0.001) feed intake, feed/gain ratio, abdominal fat percentage, and sebum percentage and lower (P = 0.001) breast muscle percentage compared with that of lean ducks. The fatty-type and lean-type ducks had similar weight gain and BW. Dietary threonine supplementation improved (P < 0.05) growth performance and increased breast muscle percentage in lean-type ducks, but it did not affect (P > 0.05) those indices in fatty-type ducks. Lean ducks had higher (P < 0.001) hepatic contents of total lipids, triglyceride, cholesterol, and plasma low-density lipoprotein cholesterol concentration, and dietary threonine supplementation decreased (P < 0.05) hepatic total lipid, cholesterol, and triglyceride contents in lean ducks, but it had no influence on hepatic lipids in fatty ducks (P > 0.05). Lean ducks had higher (P < 0.05) concentrations of monounsaturated fatty acid (MUFA), and C18-polyunsaturated fatty acid (PUFA) in the liver, PUFA in the breast muscle, and C18:3n6 and C18:3n3 in plasma and lower C20-PUFA and C22-PUFA in the liver and MUFA in plasma, compared with fatty ducks. Threonine supplementation increased PUFA, N3-PUFA, and n6-PUFA in plasma and hepatic fatty acids profiles in lean ducks (P > 0.05) but had on influence on total MUFA and total PUFA in the liver, breast muscle, and plasma in fatty ducks (P > 0.05). In conclusion, genetic selection toward meat production and threonine supplementation increases meat production and PUFA contents, which would influence eating quality, but it is benefit for human health.


Assuntos
Patos/fisiologia , Carne/análise , Seleção Genética , Treonina/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Patos/sangue , Patos/genética , Patos/crescimento & desenvolvimento , Lipídeos/análise , Fígado/química , Músculos Peitorais/química , Plasma/química , Distribuição Aleatória , Treonina/administração & dosagem
3.
PLoS One ; 15(4): e0228121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236094

RESUMO

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and ß-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.


Assuntos
Transdução de Sinal Luminoso/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Opsinas de Bastonetes/metabolismo , beta-Arrestina 1/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosforilação/fisiologia , Ligação Proteica , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes de Fusão/genética , Opsinas de Bastonetes/química , Opsinas de Bastonetes/genética , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , beta-Arrestina 1/química
4.
Mol Cell ; 78(4): 641-652.e9, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330457

RESUMO

Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.


Assuntos
ADP-Ribosilação , Proteínas de Bactérias/metabolismo , Chromobacterium/metabolismo , Poliubiquitina/metabolismo , Treonina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Proteínas de Bactérias/genética , Chromobacterium/genética , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Treonina/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação
5.
Nucleic Acids Res ; 48(9): 4811-4826, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32282918

RESUMO

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis-trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3' processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3' processing/termination that acts via Ssu72.


Assuntos
Regulação Fúngica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/genética , Fosfoproteínas Fosfatases/genética , Processamento de Terminações 3' de RNA , Proteínas de Schizosaccharomyces pombe/genética , Terminação da Transcrição Genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Deleção de Genes , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteínas Nucleares/genética , Fosfatos/metabolismo , Fosforilação , Domínios Proteicos/genética , Pirofosfatases/genética , RNA Polimerase II/genética , RNA-Seq , Regulon , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Serina/metabolismo , Treonina/metabolismo
6.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 782-791, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347072

RESUMO

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.


Assuntos
Aminobutiratos , Formiato Desidrogenases , Leucina Desidrogenase , Treonina Desidratase , Treonina , Aminobutiratos/síntese química , Bacillus thuringiensis/enzimologia , Candida/enzimologia , Escherichia coli/enzimologia , Formiato Desidrogenases/metabolismo , Leucina Desidrogenase/metabolismo , Treonina/metabolismo , Treonina Desidratase/metabolismo
7.
J Pharmacol Exp Ther ; 373(3): 370-380, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32205367

RESUMO

The pregnane X receptor (PXR), or nuclear receptor (NR) 1I2, is a ligand-activated NR superfamily member that is enriched in liver and intestine in mammals. Activation of PXR regulates the expression of genes encoding key proteins involved in drug metabolism, drug efflux, and drug transport. Recent mechanistic investigations reveal that post-translational modifications (PTMs), such as phosphorylation, play a critical role in modulating the bimodal function of PXR-mediated transrepression and transactivation of target gene transcription. Upon ligand binding, PXR undergoes a conformational change that promotes dissociation of histone deacetylase-containing multiprotein corepressor protein complexes while simultaneously favoring recruitment histone acetyl transferase-containing complexes. Here we describe a novel adenoviral vector used to deliver and recover recombinant human PXR protein from primary cultures of hepatocytes. Using liquid chromatography and tandem mass spectrometry we report here that PXR is phosphorylated at amino acid residues threonine 135 (T135) and serine 221 (S221). Biochemical analysis reveals that these two residues play an important regulatory role in the cycling of corepressor and coactivator multiprotein complexes. These data further our foundational knowledge regarding the specific role of PTMs, namely phosphorylation, in regulating the biology of PXR. Future efforts are focused on using the novel tools described here to identify additional PTMs and protein partners of PXR in primary cultures of hepatocytes, an important experimental model system. SIGNIFICANCE STATEMENT: Pregnane X receptor (PXR), or nuclear receptor 1I2, is a key master regulator of drug-inducible CYP gene expression in liver and intestine in mammals. The novel biochemical tools described in this study demonstrate for the first time that in cultures of primary hepatocytes, human PXR is phosphorylated at amino acid residues threonine 135 (T135) and serine 221 (S221). Moreover, phosphorylation of PXR promotes the transrepression of its prototypical target gene CYP3A4 through modulating its interactions with coregulatory proteins.


Assuntos
Fosforilação/fisiologia , Receptor de Pregnano X/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Camundongos , Processamento de Proteína Pós-Traducional/fisiologia , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Treonina/metabolismo
8.
Biochem Biophys Res Commun ; 525(3): 537-542, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32113680

RESUMO

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play important roles in brassinosteroid (BR)-induced antioxidant defense and enhancing the tolerance of plants to drought stress. The autophosphorylation of CCaMK is a key step for the activation of CCaMK, thus promoting substrate phosphorylation. However, how CCaMK autophosphorylation function in BR-induced antioxidant defense is not known yet. Here, seven potential autophosphorylation sites of ZmCCaMK were identified using mass spectroscopy (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) analysis. The transient gene expression analysis in maize protoplasts showed that Thr420 and Ser454 of ZmCCaMK were important for BR-induced antioxidant defense. Furthermore, Thr420 and Ser454 of ZmCCaMK were crucial for improving drought tolerance and alleviating drought induced oxidative damage of plants via overexpressing various mutant versions of ZmCCaMK in tobacco (Nicotiana tabacum). Mutations of Thr420 and Ser454 in ZmCCaMK substantially blocked the autophosphorylation and substrate phosphorylation of ZmCCaMK in vitro. Taken together, our results demonstrate that Thr420 and Ser454 of ZmCCaMK are crucial for BR-induced antioxidant defense and drought tolerance through modulating the autophosphorylation and substrate phosphorylation activities of ZmCCaMK.


Assuntos
Antioxidantes/metabolismo , Brassinosteroides/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Serina/metabolismo , Treonina/metabolismo , Zea mays/enzimologia , Adaptação Fisiológica/efeitos dos fármacos , Secas , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Tabaco/genética , Zea mays/efeitos dos fármacos
9.
Biomed Res Int ; 2020: 5246350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190670

RESUMO

Purpose: To explore the effects of depression on cardiac autonomic nerve function and related metabolic pathways, the heart rate variability (HRV) and urinary differential metabolites were detected on the college students with depression. Methods: 12 female freshmen with depression were filtered by the Beck Depression Inventory (BDI-II) and Self-rating Depression Scale (SDS). By wearing an HRV monitoring system, time domain indexes and frequency domain indexes were measured over 24 hours. Liquid chromatography-mass spectrometry (LC-MS) was used to detect their urinary differential metabolites. Differential metabolites were identified by principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). The metabolic pathways related to these differential metabolites were analyzed by the MetPA database. Results: Stress time was significantly increased, and recovery time was markedly decreased in the depression group compared with the control group (p < 0.001). Standard deviation of the normal-to-normal R interval (SDNN), root mean square of the beat-to-beat differences (RMSSD), high frequency (HF), and low frequency (LF) were decreased significantly (p < 0.001). Standard deviation of the normal-to-normal R interval (SDNN), root mean square of the beat-to-beat differences (RMSSD), high frequency (HF), and low frequency (LF) were decreased significantly (. Conclusion: Some autonomic nervous system disruption, high stress, and poor fatigue recovery were confirmed in college students with depression. The metabolic mechanism involved the disruption of coenzyme Q biosynthesis, glycine-serine-threonine metabolism, tyrosine metabolism, pyrimidine metabolism, and steroid metabolism under daily stress.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Frequência Cardíaca/fisiologia , Ubiquinona/biossíntese , Adolescente , Depressão , Fadiga , Feminino , Glicina/metabolismo , Humanos , Metabolômica , Monitorização Fisiológica , Pirimidinas/metabolismo , Serina/metabolismo , Esteroides/metabolismo , Estresse Fisiológico , Estudantes , Treonina/metabolismo , Tirosina/metabolismo , Ubiquinona/fisiologia , Urina/química , Adulto Jovem
10.
PLoS One ; 15(2): e0229256, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084204

RESUMO

Cigarette smoke (CS) contains multiple gaseous and particulate materials that can cause lung inflammation, and smoking is the major cause of chronic obstructive pulmonary disease (COPD). We sought to determine the mechanisms of how CS triggers lung inflammation. Nur77, a nuclear hormone receptor belonging to the immediate-early response gene family, controls inflammatory responses, mainly by suppressing the NF-κB signaling pathway. Because it is unknown if Nur77's anti-inflammatory role modulates COPD, we assessed if and how Nur77 expression and activity are altered in CS-induced airway inflammation. In lung tissues and bronchial epithelial cells from COPD patients, we found Nur77 was downregulated. In a murine model of CS-induced airway inflammation, CS promoted lung inflammation and also reduced Nur77 activity in wild type (WT) mice, whereas lungs of Nur77-deficient mice showed exaggerated CS-induced inflammatory responses. Our findings in in vitro studies of human airway epithelial cells complemented those in vivo data in mice, together showing that CS induced threonine-phosphorylation of Nur77, which is known to interfere with its anti-inflammatory functions. In summary, our findings point to Nur77 as an important regulator of CS-induced inflammatory responses and support the potential benefits of Nur77 activation for COPD treatment.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumaça/efeitos adversos , Tabaco/química , Animais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Inflamação/genética , Pulmão/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/patologia , Treonina/metabolismo
11.
J Anim Sci ; 98(3)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32108874

RESUMO

Threonine (Thr) requirements for immature (growing) Beagles have been determined, but little knowledge is available on Thr requirements for maintenance in mature dogs. Moreover, differences of Thr requirements among different breeds or sizes of adult dogs have not been investigated. The objective of the present study was to determine Thr requirements in adult dogs of three different breeds using the indicator amino acid oxidation (IAAO) technique. In total, 13 adult dogs were used, 4 Miniature Dachshunds (5.8 ± 0.4 kg body weight [BW]; 3 spayed and 1 neutered), 4 spayed Beagles (9.3 ± 0.6 kg BW), and 5 neutered Labrador Retrievers (30.5 ± 1.7 kg BW). Dogs were fed a Thr-deficient diet (Thr = 0.23%) and randomly allocated to receiving one of seven concentrations of Thr supplementation (final Thr concentration in experimental diets was 0.23%, 0.33%, 0.43%, 0.53%, 0.63%, 0.73%, and 0.83%; as fed basis) for 2 d. After 2 d of adaptation to the experimental diets, dogs underwent individual IAAO studies. During the IAAO studies, total daily feed was divided into 13 equal meals; at the sixth meal, dogs were fed a bolus of l-[1-13C]-Phenylalanine (Phe) (9.40 mg/kg BW), and thereafter, l-[1-13C]-Phe (2.4 mg/kg BW) was supplied with every meal. Before feeding the next experimental diet, dogs were fed a Thr-adequate basal diet for 4 d (Thr = 0.80% as fed basis) in known amounts that maintained individual dog BW. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The mean requirements for Thr, defined as the breakpoint, and the 95% confidence interval (CI) were determined using a two-phase linear regression model. For Miniature Dachshunds, the two-phase model was not significant, and Thr requirements could not be determined. Mean Thr requirements for Beagles and Labradors were 72.2 and 64.1 mg/kg BW on an as-fed basis, respectively. The requirement for Thr between these two dog breeds was not different (P > 0.10). Thus, the data for Beagles and Labradors were pooled and a mean requirement for Thr was determined at 66.9 mg/kg BW, and the 95% CI was estimated at 84.3 mg/kg BW. In conclusion, estimated Thr requirements for Beagles and Labradors did not differ, and these recommendations are higher than those suggested by NRC (2006) and AAFCO (2014) for adult dogs at maintenance.


Assuntos
Aminoácidos/metabolismo , Tamanho Corporal/fisiologia , Cães/fisiologia , Treonina/metabolismo , Ração Animal/análise , Animais , Peso Corporal , Calorimetria Indireta/veterinária , Dieta/veterinária , Feminino , Masculino , Necessidades Nutricionais , Oxirredução , Fenilalanina/metabolismo
12.
Am J Respir Cell Mol Biol ; 62(6): 719-731, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32048878

RESUMO

Decreased angiogenesis contributes to persistent pulmonary hypertension of the newborn (PPHN); mechanisms remain unclear. AMPK (5'AMP activated protein kinase) is a key regulator of cell metabolism. We investigated the hypothesis that a decrease in AMPK function leads to mitochondrial dysfunction and altered balance of notch ligands delta-like 4 (DLL4) and Jagged 1 (Jag1) to impair angiogenesis in PPHN. Studies were done in fetal lambs with PPHN induced by prenatal ductus arteriosus constriction and gestation-matched control lambs. PPHN lambs were treated with saline or AMPK agonist metformin. Angiogenesis was assessed in lungs with micro-computed tomography angiography and histology. AMPK function; expression of mitochondrial electron transport chain (ETC) complex proteins I-V, Dll4, and Jag1; mitochondrial number; and in vitro angiogenesis function were assessed in pulmonary artery endothelial cells (PAEC) from control and PPHN lambs. AMPK function was decreased in PPHN PAEC and lung sections. Expression of mitochondrial transcription factor, PGC-1α, ETC complex proteins I-V, and mitochondrial number were decreased in PPHN. In vitro angiogenesis of PAEC and capillary number and vessel volume fraction in the lung were decreased in PPHN. Expression of DLL4 was increased and Jag1 was decreased in PAEC from PPHN lambs. AMPK agonists A769662 and metformin increased the mitochondrial complex proteins and number, in vitro angiogenesis, and Jag1 levels and decreased DLL4 levels in PPHN PAEC. Infusion of metformin in vivo increased the vessel density in PPHN lungs. Decreased AMPK function contributes to impaired angiogenesis in PPHN by altered balance of notch ligands in PPHN.


Assuntos
Células Endoteliais/enzimologia , Hipertensão Pulmonar/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Patológica/enzimologia , Síndrome da Persistência do Padrão de Circulação Fetal/enzimologia , Proteínas Quinases/metabolismo , Receptores Notch/metabolismo , Animais , Animais Recém-Nascidos , Canal Arterial/embriologia , Canal Arterial/cirurgia , Transporte de Elétrons , Ativação Enzimática , Feminino , Hipertensão Pulmonar/fisiopatologia , Ligantes , Pulmão/patologia , Metformina/farmacologia , Metformina/uso terapêutico , Mitocôndrias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Síndrome da Persistência do Padrão de Circulação Fetal/tratamento farmacológico , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Síndrome da Persistência do Padrão de Circulação Fetal/fisiopatologia , Fosforilação , Gravidez , Proteínas Quinases/fisiologia , Pironas/farmacologia , Ovinos , Tiofenos/farmacologia , Treonina/metabolismo , Transfecção
13.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023819

RESUMO

The extracellular signal-regulated protein kinase 5 (ERK5) is a non-redundant mitogen-activated protein kinase (MAPK) that exhibits a unique C-terminal extension which comprises distinct structural and functional properties. Here, we sought to elucidate the significance of phosphoacceptor sites in the C-terminal transactivation domain of ERK5. We have found that Thr732 acted as a functional gatekeeper residue controlling C-terminal-mediated nuclear translocation and transcriptional enhancement. Consistently, using a non-bias quantitative mass spectrometry approach, we demonstrated that phosphorylation at Thr732 conferred selectivity for binding interactions of ERK5 with proteins related to chromatin and RNA biology, whereas a number of metabolic regulators were associated with full-length wild type ERK5. Additionally, our proteomic analysis revealed that phosphorylation of the Ser730-Glu-Thr732-Pro motif could occur independently of dual phosphorylation at Thr218-Glu-Tyr220 in the activation loop. Collectively, our results firmly establish the significance of C-terminal phosphorylation in regulating ERK5 function. The post-translational modification of ERK5 on its C-terminal tail might be of particular relevance in cancer cells where ERK5 has be found to be hyperphosphoryated.


Assuntos
Proteína Quinase 7 Ativada por Mitógeno/química , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteômica/métodos , Treonina/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Proteína Quinase 7 Ativada por Mitógeno/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais , Transcrição Genética
14.
Sci Rep ; 10(1): 3181, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081899

RESUMO

Phosphorylation of Munc18-1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndrome-related kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.


Assuntos
Síndrome de Down/enzimologia , Proteínas Munc18/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transmissão Sináptica , Animais , Sobrevivência Celular , Células HEK293 , Humanos , Camundongos , Proteínas Munc18/deficiência , Proteínas Munc18/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fases de Leitura Aberta/genética , Fosforilação , Treonina/metabolismo
15.
Nat Chem Biol ; 16(3): 351-360, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932717

RESUMO

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3's structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.


Assuntos
Fatores de Crescimento de Fibroblastos/química , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Células CHO , Cricetulus , Fatores de Crescimento de Fibroblastos/metabolismo , Glicopeptídeos/química , Glicosilação , Humanos , Isoenzimas/metabolismo , Lectinas/metabolismo , N-Acetilgalactosaminiltransferases/fisiologia , Treonina/metabolismo
16.
J Virol ; 94(8)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31969433

RESUMO

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49.IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.


Assuntos
Citomegalovirus/genética , RNA Helicases DEAD-box/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , RNA Viral/genética , Serina/metabolismo , Células HEK293 , Humanos , Mutação , Fosforilação , RNA Mensageiro/genética , Treonina/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral
17.
Proc Natl Acad Sci U S A ; 117(1): 584-594, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31843895

RESUMO

In this study, we provide critical evidence that STAT2 stability regulation plays an essential role in melanoma cell proliferation and colony growth. We found that the interaction of FBXW7 and STAT2 induced STAT2 destabilization via a ubiquitination-mediated proteasomal degradation pathway. Notably, GSK3ß-mediated STAT2 phosphorylation facilitated STAT2-FBXW7 interactions via the DNA binding domain of STAT2 and domains 1, 2, 6, and 7 of FBXW7 WD40. Importantly, the inverse correlation between protein levels of STAT2 and FBXW7 were observed not only in human melanoma cells but also in a human skin cancer tissue array. The relationship between protein levels of STAT2 and FBXW7, cell proliferation, and colony growth were similarly observed in the melanoma cell lines SK-MEL-2, -5, and -28. Moreover, STAT2 knockdown in melanoma cells suppressed melanoma cell proliferation and colony formation. These data demonstrated that FBXW7-mediated STAT2 stability regulation plays an essential role in melanoma cell proliferation and cancer growth.


Assuntos
Proteína 7 com Repetições F-Box-WD/metabolismo , Melanoma/patologia , Fator de Transcrição STAT2/metabolismo , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Estabilidade Proteica , Proteólise , Fator de Transcrição STAT2/química , Fator de Transcrição STAT2/genética , Serina/metabolismo , Transdução de Sinais , Pele/patologia , Treonina/metabolismo , Análise Serial de Tecidos , Ubiquitinação , Repetições WD40
18.
J Biol Chem ; 295(5): 1240-1260, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31857374

RESUMO

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.


Assuntos
Autofagia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Sequestossoma-1/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Cromatografia Líquida de Alta Pressão , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mutação , Quinases Relacionadas a NIMA/genética , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/genética , Espectrometria de Massas em Tandem , Treonina/metabolismo
19.
Artif Cells Nanomed Biotechnol ; 47(1): 4274-4283, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810390

RESUMO

Background: Ras-PI3K pathway aberrant activation plays an important role in the occurrence and development of osteosarcoma. This study investigated the functions of Ras-PI3K pathway specific activation on histone H2A phosphorylation at threonine 120 (H2AT120ph) in osteosarcoma cells, along with the possible internal molecular mechanisms.Methods: Cell transfection was done to alter RasG12V/Y40C, H2AT120ph and vaccinia-related kinase 1 (VRK1) expression. Then, cell viability, proliferation, migration and cell cycle distribution were assessed, respectively. qRT-PCR was utilized to measure the VRK1 and Ras-PI3K pathway downstream genes (CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16) expression. Chromatin immunoprecipitation (ChIP) was conducted to evaluate the input levels of H2AT120ph and VRK1 in the promoter regions of Ras-PI3K pathway downstream genes.Results: Ras-PI3K specific activation promoted histone H2AT120ph. H2AT120ph participated in the oncogenic functions of Ras-PI3K pathway on osteosarcoma by modulating the transcription of Ras-PI3K-targeted genes. Moreover, VRK1 contributed to the Ras-PI3K specific activation-induced up-regulation of H2AT120ph and osteosarcoma progression. Ras-PI3K pathway-specific activation-induced up-regulation of H2AT120ph was achieved by up-regulation of VRK1.Conclusions: Ras-PI3K pathway activation promoted osteosarcoma progression might be via up-regulating VRK1-mediated H2AT120ph. We proposed that VRK1 and H2AT120ph could be the potential targets for osteosarcoma diagnosis and treatment.HighlightsH2AT120ph is specifically promoted by Ras-PI3K pathway activation.H2AT120ph joins in the oncogenic effects of Ras-PI3K pathway on osteosarcoma.H2AT120ph regulates the transcription of Ras-PI3K-targeted genes.VRK1 takes part in the regulatory function of Ras-PI3K pathway on H2AT120ph.


Assuntos
Neoplasias Ósseas/patologia , Histonas/química , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas ras/metabolismo , Linhagem Celular Tumoral , Humanos , Fosforilação , Treonina/metabolismo , Transcrição Genética , Regulação para Cima
20.
Int J Mol Sci ; 21(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881804

RESUMO

Both OGT1 (O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase isoform 1) and NSL3 (nonspecific lethal protein 3) are crucial components of the MOF (males absent on the first)/NSL histone acetyltransferase complex. We previously described how global histone H4 acetylation levels were modulated by OGT1/O-GlcNAcylation-mediated NSL3 stability. However, the specific modification site of NSL3 and its molecular mechanism of protein stability remain unknown. Here, we present evidence from biochemical experiments arguing that O-GlcNAcylation of NSL3 at Thr755 is tightly associated with holoenzyme activity of the MOF/NSL complex. Using in vitro O-GlcNAc-transferase assays combined with mass spectrometry, we suppose that the residue Thr755 on NSL3 C-terminus is the major site O-GlcNAc-modified by OGT1. Importantly, O-GlcNAcylation of this site is involved in the regulation of the ubiquitin-degradation of NSL3, because this site mutation (T755A) promotes the ubiquitin-mediated degradation of NSL3. Further in-depth research found that ubiquitin conjugating enzyme E2 S (UBE2S) accelerated the degradation of NSL3 via direct binding to it. Interestingly, OGT1 and UBE2S competitively bind to NSL3, suggesting the coordination of OGT1-UBE2S in regulating NSL3 stability. Furthermore, O-GlcNAcylation of NSL3 Thr755 site regulates the histone H4 acetylation levels at lysine 5, 8, and 16, suggesting that the O-GlcNAcylation of NSL3 at Thr755 is required for maintaining the integrity and holoenzyme activity of the MOF/NSL complex. In colony formation assays, we found that the integrity of the complex impacts the proliferation of the lung carcinoma type II epithelium-like A549 cells. Taken together, our results provide new insight into the elucidation of the molecular mechanism of the MOF/NSL complex.


Assuntos
Histona Acetiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Acetilação , Células HEK293 , Histona Acetiltransferases/química , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Treonina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
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