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1.
Sci Rep ; 13(1): 1473, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36702867

RESUMO

Nitisinone (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, NTBC) is considered a potentially effective drug for the treatment of various metabolic diseases associated with disorders of L-tyrosine metabolism however, side-effects impede its widespread use. This work aimed to broaden the knowledge of the influence of NTBC and its metabolites 2-amino-4-(trifluoromethyl)benzoic acid (ATFA), 2-nitro-4-(trifluoromethyl)benzoic acid (NTFA), and cyclohexane-1,3-dione (CHD) on the catabolism of L-tyrosine and other endogenous compounds in Saccharomyces cerevisiae. Based on a targeted analysis performed by LC-ESI-MS/MS, based on multiple reaction monitoring, it was found that the dissipation kinetics of the parent compound and its metabolites are compatible with a first-order reaction mechanism. Moreover, it has been proven that formed NTBC metabolites, such as CHD, cause a decrease in L-tyrosine, L-tryptophan, and L-phenylalanine concentrations by about 34%, 59% and 51%, respectively, compared to the untreated model organism. The overall changes in the metabolism of yeast exposed to NTBC or its derivatives were evaluated by non-targeted analysis via LC-ESI-MS/MS in the ion trap scanning mode. Based on principal components analysis, a statistically significant similarity between metabolic responses of yeast treated with ATFA or NTFA was observed. These findings facilitate further studies investigating the influence of NTBC on the human body and the mechanism of its action.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Espectrometria de Massas em Tandem , Cicloexanonas/farmacologia , Cicloexanonas/uso terapêutico , Nitrobenzoatos/metabolismo , Metaboloma , Tirosina/metabolismo
2.
Cell Death Dis ; 14(1): 39, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36653376

RESUMO

The type I cGMP-dependent protein kinase (PKG I) is recognized as a tumor suppressor, but its role in EGFR regulated epithelial ovarian cancer (EOC) progression remains unclear. We evaluated the in vivo and in vitro effects of activated PKG I in EGF-induced EOC cell proliferation, migration, and invasion. The expressions of EGFR and PKG I were elevated, but the activated PKG I was decreased in EOC tissues of patients and cells lines. The addition of 8-Br-cGMP, a specific PKG I activator, attenuated the EGF-induced EOC cell proliferation, migration, and invasion in vitro. Similarly, activated PKG I also attenuated EOC progression in vivo using an EOC xenograft nude mouse model. The activated PKG I interacted with EGFR, causing increased threonine (693) phosphorylation and decreased tyrosine (1068) phosphorylation of EGFR, which resulted in disrupted EGFR-SOS1-Grb2 combination. Subsequently, the cytoplasmic phosphorylation of downstream proteins (c-Raf, MEK1/2, and ERK1/2) were declined, impeding the phosphorylated ERK1/2's nucleus translocation, and this reduction of phosphorylated tyrosine (1068) EGFR and ERK1/2 were also abolished by Rp-8-Br-cGMPS. Our results suggest that the activation of PKG I attenuates EGF-induced EOC progression, and the 8-Br-cGMP-PKG I-EGFR/MEK/ERK axis might be a potential target for EOC therapy.


Assuntos
Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas , Feminino , Animais , Camundongos , Humanos , Fosforilação , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Receptores ErbB/metabolismo , Tirosina/metabolismo
3.
Sci Rep ; 13(1): 1311, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36693869

RESUMO

Cigar variety CX-010 tobacco leaves produce localized green spots during the air-curing period, and spraying exogenous sucrose effectively alleviates the occurrence of the green spots. To investigate the alleviation effect of exogenous sucrose spraying, the total water content and the number and size of green spots on tobacco leaves were investigated during the air-curing period under four treatments; CK (pure water), T1 (0.1 M sucrose), T2 (0.2 M sucrose) and T3 (0.4 M sucrose). The results showed that the total water content of tobacco leaves showed a trend of T3 < CK < T2 < T1 in the early air-curing stage, and the number and size of green spots showed a trend of T3 < T2 < T1 < CK. All sucrose treatments alleviated the green spot phenomenon, and T3 had the fewest green spots. Thus, the tobacco leaves of the T3 and CK treatments at two air-curing stages were used to perform metabolomics analysis with nontargeted liquid chromatography‒mass spectrometry to determine the physiological mechanism. A total of 259 and 178 differentially abundant metabolites (DAMs) between T3- and CK-treated tobacco leaves were identified in the early air-curing and the end of air-curing stages, respectively. These DAMs mainly included lipid and lipid-like molecules, carbohydrates, and organic acids and their derivatives. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the T3 treatment significantly altered carbohydrate metabolism (pentose phosphate pathway, sucrose and starch metabolism and galactose metabolism) and amino acid metabolism (tyrosine metabolism and tryptophan metabolism) in air-curing tobacco leaves. Sucrose treatment alleviated green spots by altering DAMs that affected chlorophyll degradation, such as tyrosine and citric acid, to promote the normal degradation of chlorophyll.


Assuntos
Sacarose , Produtos do Tabaco , Sacarose/metabolismo , Tabaco/metabolismo , Água/metabolismo , Clorofila/metabolismo , Tirosina/metabolismo , Lipídeos/farmacologia , Folhas de Planta/metabolismo
4.
Life Sci Alliance ; 6(3)2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36535710

RESUMO

RNA 3'-end polyadenylation that marks transcripts for degradation is implicated in general stress response in Escherichia coli Yet, the mechanism and regulation of poly(A) polymerase I (PAPI) in stress response are obscure. We show that pcnB (that encodes PAPI)-null mutation widely stabilises stress response mRNAs and imparts cellular tolerance to multiple stresses, whereas PAPI ectopic expression renders cells stress-sensitive. We demonstrate that there is a substantial loss of PAPI activity on stress exposure that functionally phenocopies pcnB-null mutation stabilising target mRNAs. We identify PAPI tyrosine phosphorylation at the 202 residue (Y202) that is enormously enhanced on stress exposure. This phosphorylation inhibits PAPI polyadenylation activity under stress. Consequentially, PAPI phosphodeficient mutation (tyrosine 202 to phenylalanine, Y202F) fails to stimulate mRNA expression rendering cells stress-sensitive. Bacterial tyrosine kinase Wzc phosphorylates PAPI-Y202 residue, and that wzc-null mutation renders cells stress-sensitive. Accordingly, wzc-null mutation has no effect on stress sensitivity in the presence of pcnB-null or pcnB-Y202F mutation. We also establish that PAPI phosphorylation-dependent stress tolerance mechanism is distinct and operates downstream of the primary stress regulator RpoS.


Assuntos
Proteínas de Escherichia coli , Fosforilação , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , RNA Mensageiro/genética , Tirosina/metabolismo
5.
J Cell Biol ; 222(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36512346

RESUMO

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.


Assuntos
Proteínas Angiogênicas , Proteínas de Transporte , Proteínas de Ciclo Celular , Microtúbulos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Proteínas Angiogênicas/metabolismo
6.
Theriogenology ; 197: 159-166, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36525855

RESUMO

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 µM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 µM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 µM), and 100, 250 and 500 µM PRO, but decreased with 1000 µM PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 µM: 77.8 ± 10.6%, 100 µM: 63.7 ± 11.7%, 250 µM: 52.1 ± 12.9%, 500 µM: 37.7 ± 11.6%, 1000 µM: 33.1 ± 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 µM: 34.6%, 1000 µM: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 µM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.


Assuntos
Probucol , Sêmen , Cães , Masculino , Animais , Probucol/farmacologia , Probucol/metabolismo , Fosforilação , Sêmen/metabolismo , Motilidade Espermática/fisiologia , Espermatozoides/fisiologia , Colesterol/metabolismo , Capacitação Espermática , Reação Acrossômica , Tirosina/farmacologia , Tirosina/metabolismo
7.
ACS Synth Biol ; 12(1): 43-50, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36534785

RESUMO

Continuous directed evolution (CDE) is a powerful tool for enzyme engineering due to the depth and scale of evolutionary search that it enables. If suitably controlled and calibrated, CDE could be widely applied in plant breeding and biotechnology to improve plant enzymes ex planta. We tested this concept by evolving Arabidopsis arogenate dehydratase (AtADT2) for resistance to feedback inhibition. We used an Escherichia coli platform with a phenylalanine biosynthesis pathway reconfigured ("plantized") to mimic the plant pathway, a T7RNA polymerase-base deaminase hypermutation system (eMutaT7), and 4-fluorophenylalanine as selective agent. Selection schemes were prevalidated using a known feedback-resistant AtADT2 variant. We obtained variants that had 4-fluorophenylalanine resistance at least matching the known variant and that carried mutations in the ACT domain responsible for feedback inhibition. We conclude that ex planta CDE of plant enzymes in a microbial platform is a viable way to tailor characteristics that involve interaction with small molecules.


Assuntos
Arabidopsis , Arabidopsis/metabolismo , Escherichia coli/metabolismo , p-Fluorfenilalanina , Retroalimentação , Plantas/metabolismo , Tirosina/metabolismo
8.
J Biotechnol ; 363: 8-16, 2023 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-36566842

RESUMO

Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for l-tyrosine (l-Tyr) overproduction was attempted by pushing the carbon flux more towards l-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased l-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase l-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest l-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce l-Tyr from lignocellulosic renewable carbon sources.


Assuntos
Corynebacterium glutamicum , Engenharia Metabólica , Xilose/metabolismo , Glucose/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Tirosina/genética , Tirosina/metabolismo
9.
Microb Cell Fact ; 21(1): 278, 2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36585654

RESUMO

BACKGROUND: Melanin is a natural pigment that can be applied in different fields such as medicine, environment, pharmaceutical, and nanotechnology. Studies carried out previously showed that the melanin produced by the mel1 mutant from Aspergillus nidulans exhibits antioxidant, anti-inflammatory, and antimicrobial activities, without any cytotoxic or mutagenic effect. These results taken together suggest the potential application of melanin from A. nidulans in the pharmaceutical industry. In this context, this study aimed to evaluate the effect of factors L-tyrosine, glucose, glutamic acid, L-DOPA, and copper on melanin production by the mel1 mutant and to establish the optimal concentration of these factors to maximize melanin production. RESULTS: The results showed that L-DOPA, glucose, and copper sulfate significantly affected melanin production, where L-DOPA was the only factor that exerted a positive effect on melanin yield. Besides, the tyrosinase activity was higher in the presence of L-DOPA, considered a substrate required for enzyme activation, this would explain the increased production of melanin in this condition. After establishing the optimal concentrations of the analyzed factors, the melanin synthesis was increased by 640% compared to the previous studies. CONCLUSIONS: This study contributed to elucidating the mechanisms involved in melanin synthesis in A. nidulans as well as to determining the optimal composition of the culture medium for greater melanin production that will make it possible to scale the process for a future biotechnological application.


Assuntos
Aspergillus nidulans , Melaninas , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Levodopa , Tirosina/metabolismo , Antioxidantes
10.
Lipids Health Dis ; 21(1): 151, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36585694

RESUMO

BACKGROUND: Both hyperuricaemia and hyperlipidaemia are common metabolic diseases that are closely related to each other, and both are independent risk factors for the development of a variety of diseases. HUA combined with hyperlipidaemia increases the risk of nonalcoholic fatty liver disease and coronary heart disease. This study aimed to investigate the relationship between HUA and hyperlipidaemia and study the metabolic pathway changes in patients with HUA associated with hyperlipidaemia using metabolomics. METHODS: This was a case‒control study. The prevalence of hyperlipidaemia in HUA patients in the physical examination population of Tianjin Union Medical Centre in 2018 was investigated. Metabolomics analysis was performed on 308 HUA patients and 100 normal controls using Orbitrap mass spectrometry. A further metabolomics study of 30 asymptomatic HUA patients, 30 HUA patients with hyperlipidaemia, and 30 age-and sex-matched healthy controls was conducted. Differential metabolites were obtained from the three groups by orthogonal partial least-squares discrimination analysis, and relevant metabolic pathways changes were analysed using MetaboAnalyst 5.0 software. RESULTS: The prevalence of hyperlipidaemia in HUA patients was 69.3%. Metabolomic analysis found that compared with the control group, 33 differential metabolites, including arachidonic acid, alanine, aspartate, phenylalanine and tyrosine, were identified in asymptomatic HUA patients. Pathway analysis showed that these changes were mainly related to 3 metabolic pathways, including the alanine, aspartate and glutamate metabolism pathway. Thirty-eight differential metabolites, including linoleic acid, serine, glutamate, and tyrosine, were identified in HUA patients with hyperlipidaemia. Pathway analysis showed that they were mainly related to 7 metabolic pathways, including the linoleic acid metabolism pathway, phenylalanine, tyrosine and tryptophan biosynthesis pathway, and glycine, serine and threonine metabolism pathway. CONCLUSIONS: Compared to the general population, the HUA population had a higher incidence of hyperlipidaemia. HUA can cause hyperlipidaemia. by affecting the metabolic pathways of linoleic acid metabolism and alanine, aspartate and glutamate metabolism. Fatty liver is closely associated with changes in the biosynthesis pathway of pahenylalanine, tyrosine, and tryptophan in HUA patients with hyperlipidaemia. Changes in the glycine, serine and threonine metabolism pathway in HUA patients with hyperlipidaemia may lead to chronic kidney disease.


Assuntos
Hiperlipidemias , Hiperuricemia , Doenças Metabólicas , Humanos , Triptofano/metabolismo , Ácido Aspártico/metabolismo , Estudos de Casos e Controles , Hiperlipidemias/complicações , Ácido Linoleico , Espectrometria de Massas , Redes e Vias Metabólicas , Tirosina/metabolismo , Fenilalanina/metabolismo , Treonina/metabolismo , Alanina/metabolismo , Glicina/metabolismo , Serina/metabolismo , Biomarcadores/metabolismo
11.
Biomolecules ; 12(12)2022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36551282

RESUMO

By denaturing proteins and promoting the formation of multiprotein complexes, protein phosphorylation has important effects on the activity of protein functional molecules and cell signaling. The regulation of protein phosphorylation allows microbes to respond rapidly and reversibly to specific environmental stimuli or niches, which is closely related to the molecular mechanisms of bacterial drug resistance. Accurate prediction of phosphorylation sites (p-site) of prokaryotes can contribute to addressing bacterial resistance and providing new perspectives for developing novel antibacterial drugs. Most existing studies focus on human phosphorylation sites, while tools targeting phosphorylation site identification of prokaryotic proteins are still relatively scarce. This study designs a capsule network-based prediction technique for p-site in prokaryotes. To address the poor scalability and unreliability of dynamic routing processes in the output space of capsule networks, a more reliable way is introduced to learn the consistency between capsules. We incorporate a self-attention mechanism into the routing algorithm to capture the global information of the capsule, reducing the computational effort while enriching the representation capability of the capsule. Aiming at the weak robustness of the model, EcapsP improves the prediction accuracy and stability by introducing shortcuts and unconditional reconfiguration. In addition, the study compares and analyzes the prediction performance based on word vectors, physicochemical properties, and mixing characteristics in predicting serine (Ser/S), threonine (Thr/T), and tyrosine (Tyr/Y) p-site. The comprehensive experimental results show that the accuracy of the developed technique is close to 70% for the identification of the three phosphorylation sites in prokaryotes. Importantly, in side-by-side comparisons with other state-of-the-art predictors, our method improves the Matthews correlation coefficient (MCC) by approximately 7%. The results demonstrate the superiority of EcapsP in terms of high performance and reliability.


Assuntos
Células Procarióticas , Proteínas , Humanos , Fosforilação , Reprodutibilidade dos Testes , Proteínas/metabolismo , Tirosina/metabolismo
12.
Int J Mol Sci ; 23(24)2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36555586

RESUMO

SH2 (Src Homology 2) domains are among the best characterized and most studied protein-protein interaction (PPIs) modules able to bind and recognize sequences presenting a phosphorylated tyrosine. This post-translational modification is a key regulator of a plethora of physiological and molecular pathways in the eukaryotic cell, so SH2 domains possess a fundamental role in cell signaling. Consequently, several pathologies arise from the dysregulation of such SH2-domains mediated PPIs. In this review, we recapitulate the current knowledge about the structural, folding stability, and binding properties of SH2 domains and their roles in molecular pathways and pathogenesis. Moreover, we focus attention on the different strategies employed to modulate/inhibit SH2 domains binding. Altogether, the information gathered points to evidence that pharmacological interest in SH2 domains is highly strategic to developing new therapeutics. Moreover, a deeper understanding of the molecular determinants of the thermodynamic stability as well as of the binding properties of SH2 domains appears to be fundamental in order to improve the possibility of preventing their dysregulated interactions.


Assuntos
Tirosina , Domínios de Homologia de src , Fosfotirosina/metabolismo , Tirosina/metabolismo , Transdução de Sinais , Ligação Proteica , Sítios de Ligação
13.
Cells ; 11(23)2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36497030

RESUMO

The immunophilin FKBP51 forms heterocomplexes with molecular chaperones, protein-kinases, protein-phosphatases, autophagy-related factors, and transcription factors. Like most scaffold proteins, FKBP51 can use a simple tethering mechanism to favor the efficiency of interactions with partner molecules, but it can also exert more complex allosteric controls over client factors, the immunophilin itself being a putative regulation target. One of the simplest strategies for regulating pathways and subcellular localization of proteins is phosphorylation. In this study, it is shown that scaffold immunophilin FKBP51 is resolved by resolutive electrophoresis in various phosphorylated isoforms. This was evidenced by their reactivity with specific anti-phosphoamino acid antibodies and their fade-out by treatment with alkaline phosphatase. Interestingly, stress situations such as exposure to oxidants or in vivo fasting favors FKBP51 translocation from mitochondria to the nucleus. While fasting involves phosphothreonine residues, oxidative stress involves tyrosine residues. Molecular modeling predicts the existence of potential targets located at the FK1 domain of the immunophilin. Thus, oxidative stress favors FKBP51 dephosphorylation and protein degradation by the proteasome, whereas FK506 binding protects the persistence of the post-translational modification in tyrosine, leading to FKBP51 stability under oxidative conditions. Therefore, FKBP51 is revealed as a phosphoprotein that undergoes differential phosphorylations according to the stimulus.


Assuntos
Fosfoproteínas , Proteínas de Ligação a Tacrolimo , Humanos , Fosfoproteínas/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Peptidilprolil Isomerase/metabolismo , Tirosina/metabolismo
14.
Cells ; 11(24)2022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36552734

RESUMO

Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl-deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.


Assuntos
Esclerose Amiotrófica Lateral , Proteínas Proto-Oncogênicas c-abl , Humanos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neuroblastoma , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Tirosina/metabolismo
15.
Front Immunol ; 13: 1054920, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569841

RESUMO

The LAT transmembrane adaptor is essential to transduce intracellular signals triggered by the TCR. Phosphorylation of its four C-terminal tyrosine residues (136, 175, 195, and 235 in mouse LAT) recruits several proteins resulting in the assembly of the LAT signalosome. Among those tyrosine residues, the one found at position 136 of mouse LAT plays a critical role for T cell development and activation. The kinetics of phosphorylation of this residue is delayed as compared to the three other C-terminal tyrosines due to a conserved glycine residue found at position 135. Mutation of this glycine into an aspartate residue (denoted LATG135D) increased TCR signaling and altered antigen recognition in human Jurkat T cells and ex vivo mouse T cells. Here, using a strain of LATG135D knockin mice, we showed that the LATG135D mutation modifies thymic development, causing an increase in the percentage of CD4+CD8+ double-positive cells, and a reduction in the percentage of CD4+ and CD8+ single-positive cells. Interestingly, the LATG135D mutation alters thymic development even in a heterozygous state. In the periphery, the LATG135D mutation reduces the percentage of CD8+ T cells and results in a small increment of γδ T cells. Remarkably, the LATG135D mutation dramatically increases the percentage of central memory CD8+ T cells. Finally, analysis of the proliferation and activation of T lymphocytes shows increased responses of T cells from mutant mice. Altogether, our results reinforce the view that the residue preceding Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Tirosina , Camundongos , Animais , Humanos , Tirosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Mutação
16.
Front Immunol ; 13: 1072702, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569848

RESUMO

The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody's binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed.


Assuntos
Regiões Determinantes de Complementaridade , Cadeias Pesadas de Imunoglobulinas , Regiões Determinantes de Complementaridade/genética , Cadeias Pesadas de Imunoglobulinas/genética , Antígenos , Complexo de Golgi/metabolismo , Tirosina/metabolismo
17.
Oxid Med Cell Longev ; 2022: 9233749, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36406767

RESUMO

Mitophagy and oxidative stress play important roles in Parkinson's disease (PD). Dysregulated mitophagy exacerbates mitochondrial oxidative damage; however, the regulatory mechanism of mitophagy is unclear. Here, we provide a potential mechanistic link between c-Abl, a nonreceptor tyrosine kinase, and mitophagy in PD progression. We found that c-Abl activation reduces the interaction of prohibitin 2 (PHB2) and microtubule-associated protein 1 light chain 3 (LC3) and decreases the expressive level of antioxidative stress proteins, including nuclear factor erythroid 2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO-1), and the antioxidant enzyme heme oxygenase-1 (HO-1) in 1-methyl-4-phenylpyridinium- (MPP+-) lesioned SH-SY5Y cells. Importantly, we found that MPP+ can increase the expression of phosphorylated proteins at the tyrosine site of PHB2 and the interaction of c-Abl with PHB2. We showed for the first time that PHB2 by changing tyrosine (Y) to aspartate (D) at site 121 resulted in impaired binding of PHB2 and LC3 in vitro. Moreover, silencing of PHB2 can decrease the interaction of PHB2 and LC3 and exacerbate the loss of dopaminergic neurons. We also found that STI 571, a c-Abl family kinase inhibitor, can decrease dopaminergic neuron damage and ameliorate MPTP-induced behavioral deficits in PD mice. Taken together, our findings highlight a novel molecular mechanism for aberrant PHB2 phosphorylation as an inhibitor of c-Abl activity and suggest that c-Abl and PHB2 are potential therapeutic targets for the treatment of individuals with PD. However, these results need to be further validated in PHB2 Y121D mice.


Assuntos
Neuroblastoma , Doença de Parkinson , Animais , Humanos , Camundongos , Mitofagia , Fosforilação , Doença de Parkinson/tratamento farmacológico , TYK2 Quinase/metabolismo , TYK2 Quinase/uso terapêutico , Proibitinas , 1-Metil-4-fenilpiridínio , Tirosina/metabolismo
18.
Commun Biol ; 5(1): 1251, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36380187

RESUMO

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.


Assuntos
Cardiomiopatia Hipertrófica , Proteoma , Camundongos , Animais , Proteoma/metabolismo , Fosforilação , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomegalia , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo
19.
Anim Sci J ; 93(1): e13777, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36342023

RESUMO

This study aimed to verify the effects of polyvinyl alcohol (PVA) and bovine serum albumin (BSA) on the induction of full-type hyperactivation in boar spermatozoa treated with a cyclic AMP analog (cBiMPS). Washed spermatozoa were treated with cBiMPS (100 µM) for 180 min. As shown in the assessment of sperm motility, PVA (0.05%-0.4%) significantly promoted the induction of full-type hyperactivation, whereas BSA (0.025%-0.4%) did not affect the induction. In comparative experiments, BSA (0.4%) effectively promoted the induction of full-type hyperactivation in bovine spermatozoa treated with cBiMPS, calyculin A (a protein phosphatase inhibitor), and digoxin (a Na+ /K+ -ATPase inhibitor), while PVA (0.1%) did not affect the induction. Western blotting showed that protein tyrosine phosphorylation states of >50 kDa sperm proteins were effectively enhanced by treatment with cBiMPS in the PVA/BSA-free medium and not affected by the addition of PVA (0.1%). The assessment of plasma membrane integrity indicated that BSA (0.4%) significantly decreased spermatozoa with intact plasma membranes. These results indicate that PVA (0.1%) promotes the induction of full-type hyperactivation and does not influence the protein tyrosine phosphorylation states in boar cBiMPS-treated spermatozoa. They also suggest that BSA should not be added to medium containing cBiMPS for boar spermatozoa.


Assuntos
AMP Cíclico , Motilidade Espermática , Suínos , Masculino , Animais , AMP Cíclico/farmacologia , Álcool de Polivinil/farmacologia , Álcool de Polivinil/metabolismo , Soroalbumina Bovina/farmacologia , Soroalbumina Bovina/metabolismo , Sêmen/metabolismo , Espermatozoides/fisiologia , Tirosina/metabolismo
20.
Cells ; 11(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36359823

RESUMO

Preeclampsia is a pregnancy-specific disorder involving placental abnormalities. Elevated placental Sialic acid immunoglobulin-like lectin (Siglec)-6 expression has been correlated with preeclampsia. Siglec-6 is a transmembrane receptor, expressed predominantly by the trophoblast cells in the human placenta. It interacts with sialyl glycans such as sialyl-TN glycans as well as binds leptin. Siglec-6 overexpression has been shown to influence proliferation, apoptosis, and invasion in the trophoblast (BeWo) cell model. However, there is no direct evidence that Siglec-6 plays a role in preeclampsia pathogenesis and its signaling potential is still largely unexplored. Siglec-6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic tail suggesting a signaling function. Site-directed mutagenesis and transfection were employed to create a series of Siglec-6 expressing HTR-8/SVneo trophoblastic cell lines with mutations in specific functional residues to explore the signaling potential of Siglec-6. Co-immunoprecipitation and inhibitory assays were utilized to investigate the association of Src-kinases and SH-2 domain-containing phosphatases with Siglec-6. In this study, we show that Siglec-6 is phosphorylated at ITIM and ITIM-like domains by Src family kinases. Phosphorylation of both ITIM and ITIM-like motifs is essential for the recruitment of phosphatases like Src homology region 2 containing protein tyrosine phosphatase 2 (SHP-2), which has downstream signaling capabilities. These findings suggest Siglec-6 as a signaling molecule in human trophoblasts. Further investigation is warranted to determine which signaling pathways are activated downstream to SHP-2 recruitment and how overexpression of Siglec-6 in preeclamptic placentas impacts pathogenesis.


Assuntos
Lectinas , Pré-Eclâmpsia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Quinases da Família src , Feminino , Humanos , Gravidez , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Fosforilação , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Quinases da Família src/metabolismo , Tirosina/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Lectinas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
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