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1.
J Agric Food Chem ; 69(46): 13895-13903, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34757739

RESUMO

Bio-based propionate is widely welcome in the food additive industry. The current anaerobic process by Propionibacteria endures low titers and a long fermentation time. In this study, a new route for propionate production from l-threonine was designed. 2-Ketobutyrate, deaminated from l-threonine, is cleaved into propionaldehyde and CO2 and then be oxidized into propionic acid, which is neutralized by ammonia released from the first deamination step. This CoA-independent pathway with only CO2 as a byproduct boosts propionate production from l-threonine with high productivity and purity. The key enzyme for 2-ketobutyrate decarboxylation was selected, and its expression was optimized. The engineered Pseudomonas putida strain, harboring 2-ketoisovalerate decarboxylase from Lactococcus lactis could produce 580 mM (43 g/L) pure propionic acid from 600 mM l-threonine in 24 h in the batch biotransformation process. Furthermore, a high titer of 62 g/L propionic acid with a productivity of 1.07 g/L/h and a molar yield of >0.98 was achieved in the fed-batch pattern. Finally, an efficient sequential fermentation-biotransformation process was demonstrated to produce propionate directly from the fermentation broth containing l-threonine, which further reduces the costs since no l-threonine purification step is required.


Assuntos
Propionatos , Pseudomonas putida , Biotransformação , Fermentação , Pseudomonas putida/metabolismo , Treonina/metabolismo
2.
Anticancer Res ; 41(10): 4789-4799, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34593428

RESUMO

BACKGROUND/AIM: This study analysed threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) expression and investigated whether pSmad2/3L-Thr is related to the transition from human colorectal adenoma (CRA) to carcinoma (CRC). MATERIALS AND METHODS: Immunofluorescent staining was performed forß-catenin, p53, CDK4, Ki67, Sox9, aldehyde dehydrogenase (ALDH) 1, and pSmad2/3L-Thr. RESULTS: We analysed specimens of diffuse p53-positive CRCs arising from p53-negative CRAs. Percentage of p53, nuclear ß-catenin, Ki67, CDK4, and pSmad2/3L-Thr-positive cells at the site of CRCs was significantly higher than that at the site of CRAs. At the site of normal colorectal mucosae, few epithelial cells were stained positively for pSmad2/3L-Thr. At the site of CRCs, pSmad2/3L-Thr-positive cells showed co-localization with p53, nuclear ß-catenin, and ALDH1. At any site, pSmad2/3L-Thr-positive cells showed co-localization with CDK4. CONCLUSION: pSmad2/3L-Thr correlates with human CRC carcinogenesis, and pSmad2/3L-Thr-positive cells show human colorectal stem cell-like and cancer stem cell characteristics.


Assuntos
Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Neoplasias Colorretais/patologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células-Tronco Neoplásicas/patologia , Fosforilação , Treonina/metabolismo , Proteína Supressora de Tumor p53/metabolismo
3.
Nat Commun ; 12(1): 5650, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34561442

RESUMO

Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.


Assuntos
Enzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Processamento de Proteína Pós-Traducional , Sítios de Ligação/genética , Enzimas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Espectrometria de Massas/métodos , Metabolômica/métodos , Mutação , Fosforilação , Proteoma/metabolismo , Proteômica/métodos , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismo
4.
Nutrients ; 13(8)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34444752

RESUMO

Threonine (Thr), an essential amino acid for animals and the limiting amino acid in swine and poultry diets, which plays a vital role in the modulation of nutritional metabolism, macromolecular biosynthesis, and gut homeostasis. Current evidence supports that the supplementation of Thr leads to benefits in terms of energy metabolism. Threonine is not only an important component of gastrointestinal mucin, but also acts as a nutritional modulator that influences the intestinal immune system via complex signaling networks, particularly mitogen-activated protein kinase (MAPK) and the target of the rapamycin (TOR) signal pathway. Threonine is also recognized as an indispensable nutrient for cell growth and proliferation. Hence, optimization of Thr requirement may exert a favorable impact on the factors linked to health and diseases in animals. This review focuses on the latest reports of Thr in metabolic pathways and nutritional regulation, as well as the relationship between Thr and relevant physiological functions.


Assuntos
Metabolismo Energético , Estado Nutricional , Treonina/metabolismo , Aminoácidos/metabolismo , Animais , Dieta , Trato Gastrointestinal/metabolismo , Redes e Vias Metabólicas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucinas/metabolismo , Sirolimo , Células-Tronco , Suínos
5.
Int J Mol Sci ; 22(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34299293

RESUMO

Brassinosteroids (BRs) are growth-promoting phytohormones that can efficiently function by exogenous application at micromolar concentrations or by endogenous fine-tuning of BR-related gene expression, thus, precisely controlling BR signal strength is a key factor in exploring the agricultural potential of BRs. BRASSINOSTEROID INSENSITIVE1 (BRI1), a BR receptor, is the rate-limiting enzyme in BR signal transduction, and the phosphorylation of each phosphorylation site of SlBRI1 has a distinct effect on BR signal strength and botanic characteristics. We recently demonstrated that modifying the phosphorylation sites of tomato SlBRI1 could improve the agronomic traits of tomato to different extents; however, the associated agronomic potential of SlBRI1 phosphorylation sites in tomato has not been fully exploited. In this research, the biological functions of the phosphorylation site threonine-825 (Thr-825) of SlBRI1 in tomato were investigated. Phenotypic analysis showed that, compared with a tomato line harboring SlBRI1, transgenic tomato lines expressing SlBRI1 with a nonphosphorylated Thr-825 (T825A) exhibited a larger plant size due to a larger cell size and higher yield, including a greater plant height, thicker stems, longer internodal lengths, greater plant expansion, a heavier fruit weight, and larger fruits. Molecular analyses further indicated that the autophosphorylation level of SlBRI1, BR signaling, and gibberellic acid (GA) signaling were elevated when SlBRI1 was dephosphorylated at Thr-825. Taken together, the results demonstrated that dephosphorylation of Thr-825 can enhance the functions of SlBRI1 in BR signaling, which subsequently activates and cooperates with GA signaling to stimulate cell elongation and then leads to larger plants and higher yields per plant. These results also highlight the agricultural potential of SlBRI1 phosphorylation sites for breeding high-yielding tomato varieties through precise control of BR signaling.


Assuntos
Brassinosteroides/metabolismo , Lycopersicon esculentum/genética , Proteínas Serina-Treonina Quinases/genética , Tamanho Celular , Frutas/metabolismo , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Treonina/metabolismo
6.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299370

RESUMO

Primary cilia are commonly found on most quiescent, terminally differentiated cells and play a major role in the regulation of the cell cycle, cell motility, sensing, and cell-cell communication. Alterations in ciliogenesis and cilia maintenance are causative of several human diseases, collectively known as ciliopathies. A key determinant of primary cilia is the histone deacetylase HDAC6, which regulates their length and resorption and whose distribution is regulated by the death inducer-obliterator 3 (Dido3). Here, we report that the atypical protein kinase Haspin is a key regulator of cilia dynamics. Cells defective in Haspin activity exhibit longer primary cilia and a strong delay in cilia resorption upon cell cycle reentry. We show that Haspin is active in quiescent cells, where it phosphorylates threonine 3 of histone H3, a known mitotic Haspin substrate. Forcing Dido3 detachment from the chromatin prevents Haspin inhibition from impacting cilia dynamics, suggesting that Haspin activity is required for the relocalization of Dido3-HDAC6 to the basal body. Exploiting the zebrafish model, we confirmed the physiological relevance of this mechanism. Our observations shed light on a novel player, Haspin, in the mechanisms that govern the determination of cilia length and the homeostasis of mature cilia.


Assuntos
Cílios/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Treonina/metabolismo , Animais , Ciclo Celular/fisiologia , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Humanos , Peixe-Zebra
7.
Hamostaseologie ; 41(3): 206-216, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34192779

RESUMO

Comprehensive proteomic analyses of human and murine platelets established an extraordinary intracellular repertoire of signaling components, which control crucial functions. The spectrum of platelet serine/threonine protein kinases (more than 100) includes the AGC family (protein kinase A, G, C [PKA, PKG, PKC]), the mitogen-activated protein kinases (MAPKs), and others. PKA and PKG have multiple significantly overlapping substrates in human platelets, which possibly affect functions with clear "signaling nodes" of regulation by multiple protein kinases/phosphatases. Signaling nodes are intracellular Ca2+ stores, the contractile system (myosin light chains), and other signaling components such as G-proteins, protein kinases, and protein phosphatases. An example for this fine-tuning is the tyrosine kinase Syk, a crucial component of platelet activation, which is controlled by several serine/threonine and tyrosine protein kinases as well as phosphatases. Other protein kinases including PKA/PKG modulate protein phosphatase 2A, which may be a master regulator of MAPK signaling in human platelets. Protein kinases and in particular MAPKs are targeted by an increasing number of clinically used inhibitors. However, the precise regulation and fine-tuning of these protein kinases and their effects on other signaling components in platelets are only superficially understood-just the beginning. However, promising future approaches are in sight.


Assuntos
Plaquetas/efeitos dos fármacos , Fosfoproteínas Fosfatases/farmacologia , Proteínas Quinases/farmacologia , Serina/metabolismo , Treonina/metabolismo , Animais , Plaquetas/metabolismo , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Animais , Cadeias Leves de Miosina/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Proteômica , Transdução de Sinais , Quinase Syk/metabolismo
8.
Aging (Albany NY) ; 13(10): 13739-13763, 2021 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-34023818

RESUMO

ETS1 - an evolutionarily conserved transcription factor involved in the regulation of a number of cellular processes - is overexpressed in several malignancies, including ovarian cancer. Most studies on ETS1 expression have been focused on the transcriptional and RNA levels, with post-translational control mechanisms remaining relatively unexplored in the pathogenesis of malignancies. Here, we show that ETS1 forms a complex with glycogen synthase kinase-3ß (GSK3ß). Specifically, GSK3ß-mediated phosphorylation of ETS1 at threonine 265 and serine 269 promoted protein stability, induced the transcriptional activation of matrix metalloproteinase (MMP)-9, and increased cell migration. In vivo experiments revealed that a GSK3ß inhibitor was able to suppress both endogenous ETS1 expression and induction of MMP-9 expression. Upon generation of a specific antibody against phosphorylated ETS1, we demonstrated that phospho-ETS1 immunohistochemical expression in ovarian cancer specimens was correlated with that of MMP-9. Notably, the cumulative overall survival of patients with low phospho-ETS1 histoscores was significantly longer than that of those showing higher scores. We conclude that the GSK3ß/ETS1/MMP-9 axis may regulate the biological aggressiveness of ovarian cancer and can serve as a prognostic factor in patients with this malignancy.


Assuntos
Progressão da Doença , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Proto-Oncogênica c-ets-1/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Estadiamento de Neoplasias , Fosforilação , Ligação Proteica , Estabilidade Proteica , Proteína Proto-Oncogênica c-ets-1/química , Proteína Proto-Oncogênica c-ets-1/genética , Serina/metabolismo , Especificidade por Substrato , Treonina/metabolismo
9.
Mol Cell ; 81(11): 2460-2476.e11, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33974913

RESUMO

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.


Assuntos
Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
10.
Sci Rep ; 11(1): 11272, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050207

RESUMO

O-GlcNAcylation, an energy-sensitive posttranslational modification, can regulate the activity of endothelial nitric oxide synthase (eNOS). Previous studies found that Thr866 is the key site for low-glucose-mediated regulation of eNOS O-GlcNAc. However, it is not known whether this activity functions through the Thr866 site concomitant with other physical and chemical factors. Therefore, we first explored the effects of physical and chemical factors on eNOS O-GlcNAc and its Thr866 site. In this study, hypertonic stress, hyperthermia and hydrogen peroxide all increased the expression levels of eNOS O-GlcNAc, whereas hypoxia and high levels of alcohol had no effect. on the expression levels of eNOS O-GlcNAc; by contrast, low pH led to a decrease in eNOS O-GlcNAc levels. Notably, eNOS O-GlcNAc protein levels were unchanged after Thr866 site mutation only under hypertonic conditions, suggesting that hypertonic stress may act through the Thr866 site. Upon exploring the mechanism of hypertonic stress on eNOS O-GlcNAc activity and function, we found that hypertonic stress can upregulate the expression of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), which is dependent on AMPK. When AMPK was knocked out, the upregulation of OGT expression and increased O-GlcNAc modifications induced by hypertonic stress were reversed.


Assuntos
Acetilglucosamina/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Pressão Osmótica/fisiologia , Acetilglucosamina/fisiologia , Animais , Bovinos , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Glicosilação , Células HEK293 , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Treonina/metabolismo
11.
Food Funct ; 12(13): 5821-5836, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34047325

RESUMO

The use of antimicrobial peptide (AMP), found in all forms of life and playing a pivotal role in the innate immune system, has been developed as a new strategy for maintaining intestinal health and reducing antibiotic usage due to its ability to resist pathogens and commensal microbes. The current study investigated the effects of l-threonine on ß-defensin expression, the intestinal mucosal barrier and inflammatory cytokine expression in porcine intestinal epithelial cell lines (IPEC-J2). The results revealed that in IPEC-J2 cells, l-threonine significantly increased the expression of ß-defensin (including pBD-1, pBD-2, and pBD-3) in a dose- and time-dependent manner (P < 0.05). By using different concentrations and treatment times of l-threonine, the results showed that the expression of ß-defensin was upregulated to the greatest extent in IPEC-J2 cells cultured with 1 mM l-threonine for 24 h. Although the mRNA expression levels of ß-defensins were markedly increased (P < 0.05), there was relatively little inducible pBD-1, pBD-2 and pBD-3 mRNA expression at the sub-confluent and confluent densities in comparison with post-confluent densities. Furthermore, we found that l-threonine enhanced the ß-defensin expression by suppressing the expression of SIRT1, which increased acetylated p65 expression, and activating the NF-κB signaling pathway, which induced the translocation of p65 from the cytoplasm to the nucleus. In addition, l-threonine significantly prevented LPS-induced intestinal mucosal barrier damage by attenuating the decreasing tendency of the mRNA expression of Mucin1 and Mucin2 (P < 0.05). Simultaneously, l-threonine enhanced the expression of ß-defensins upon LPS challenge in IPEC-J2 cells (P < 0.05). l-Threonine obviously decreased the mRNA expression of inflammatory cytokines compared to that in untreated cells (P < 0.05). In conclusion, l-threonine can upregulate ß-defensin expression and reduce inflammatory cytokine expression in IPEC-J2 cells; meanwhile, l-threonine alleviates LPS-induced intestinal mucosal barrier damage in porcine intestinal epithelial cells. The l-threonine-mediated modulation of endogenous defensin expression may be a promising approach to reduce antibiotic use, enhance disease resistance and intestinal health in animals.


Assuntos
Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Treonina/farmacologia , Regulação para Cima/efeitos dos fármacos , beta-Defensinas/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , NF-kappa B/genética , Proteínas de Neoplasias , Proteínas de Transporte Nucleocitoplasmático , RNA Mensageiro/metabolismo , Sirtuína 1/genética , Suínos , Treonina/metabolismo , beta-Defensinas/genética
12.
J Alzheimers Dis ; 81(2): 769-785, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33814431

RESUMO

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are rare neurodegenerative disorders that affect animals and humans. Bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeld-Jakob Disease (CJD) in humans belong to this group. The causative agent of TSEs is called "prion", which corresponds to a pathological form (PrPSc) of a normal cellular protein (PrPC) expressed in nerve cells. PrPSc is resistant to degradation and can induce abnormal folding of PrPC, and TSEs are characterized by extensive spongiosis and gliosis and the presence of PrPSc amyloid plaques. CJD presents initially with clinical symptoms similar to Alzheimer's disease (AD). In AD, tau aggregates and amyloid-ß protein plaques are associated with memory loss and cognitive impairment in patients. OBJECTIVE: In this work, we study the role of tau and its relationship with PrPSc plaques in CJD. METHODS: Multiple immunostainings with specific antibodies were carried out and analyzed by confocal microscopy. RESULTS: We found increased expression of the glial fibrillary acidic protein (GFAP) and matrix metalloproteinase (MMP-9), and an exacerbated apoptosis in the granular layer in cases with prion disease. In these cases, tau protein phosphorylated at Thr-231 was overexpressed in the axons and dendrites of Purkinje cells and the extensions of parallel fibers in the cerebellum. CONCLUSION: We conclude that phosphorylation of tau may be a response to a toxic and inflammatory environment generated by the pathological form of prion.


Assuntos
Cerebelo/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Doenças Priônicas/patologia , Proteínas tau/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Encefalopatias/metabolismo , Encefalopatias/patologia , Bovinos , Cerebelo/patologia , Síndrome de Creutzfeldt-Jakob/metabolismo , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Priônicas/metabolismo , Treonina/metabolismo
13.
J Biol Chem ; 296: 100551, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33744286

RESUMO

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rho-associated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention.


Assuntos
Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Glucocorticoides/metabolismo , Treonina/metabolismo , Proteínas 14-3-3/genética , Células HEK293 , Humanos , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Receptores de Glucocorticoides/genética , Treonina/genética , Ativação Transcricional
14.
Nat Commun ; 12(1): 1899, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771996

RESUMO

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.


Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Treonina/metabolismo , Motivos de Aminoácidos/genética , Animais , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Ativação Enzimática , Feminino , Humanos , Oócitos/metabolismo , Fosforilação , Prolina/genética , Prolina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Serina/genética , Serina/metabolismo , Treonina/genética , Xenopus laevis
15.
Angew Chem Int Ed Engl ; 60(15): 8460-8465, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33586286

RESUMO

Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post-translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post-translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward-prenylated on a threonine residue, representing an unprecedented post-translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O-prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology.


Assuntos
Proteínas de Bactérias/química , Dimetilaliltranstransferase/química , Peptídeos Cíclicos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Dimetilaliltranstransferase/isolamento & purificação , Dimetilaliltranstransferase/metabolismo , Estrutura Molecular , Peptídeos Cíclicos/metabolismo , Treonina/química , Treonina/metabolismo
16.
J Basic Microbiol ; 61(4): 339-350, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33570201

RESUMO

Environment and food contamination with cadmium (Cd) can cause serious toxicity, posing a severe threat to agricultural production and human health. However, how amino acids contribute to defenses against oxidative stress caused by Cd in cells is not fully understood. As a model eukaryote with a relatively clear genetic background, Saccharomyces cerevisiae has been commonly used in Cd toxicity research. To gain insight into Cd toxicity and cell defenses against it, 20 amino acids were screened for protective roles against Cd stress in S. cerevisiae. The results showed that threonine (Thr, T) had the strongest protective effect against Cd-induced mortality and membrane damage in the cells. Compared to the antioxidant vitamin C (VC), Thr exhibited a higher efficacy in restoring the superoxide dismutase (SOD) activity that was inhibited by Cd but not by H2 O2 in vivo. Thr exhibited evident DPPH (2,2-diphenyl-1-picrylhydrazyl) activity but weak ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-9 sulfonic acid)) scavenging activity, giving it a weaker effect against Cd-induced lipid peroxidation and superoxide radical O2- , compared to VC. More importantly, compared to the chelating agent EDTA, Thr showed stronger chelation of Cd, giving it a stronger protective effect on SOD against Cd than VC in vitro. The results of the in vivo and in vitro experiments revealed that the role Thr plays in cell defenses against Cd may be attributed to its protection of the SOD enzyme, predominantly through the preferential chelation of Cd. Our results provide insights into the protective mechanisms of amino acid Thr that ameliorate Cd toxicity and suggest that a supplement of Thr might help to reduce Cd-induced oxidative damage.


Assuntos
Cádmio/toxicidade , Saccharomyces cerevisiae/metabolismo , Treonina/farmacologia , Antioxidantes/metabolismo , Benzotiazóis , Catalase/metabolismo , Sequestradores de Radicais Livres , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácidos Sulfônicos , Superóxido Dismutase/metabolismo , Treonina/metabolismo
17.
Int J Mol Sci ; 22(3)2021 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-33498635

RESUMO

The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Anticorpos/metabolismo , Ativação Enzimática , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Mutação , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/imunologia
18.
Nucleic Acids Res ; 49(6): 3461-3489, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33398329

RESUMO

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.


Assuntos
Autoantígenos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Biossíntese de Proteínas , Ribonucleoproteínas/metabolismo , Motivos de Aminoácidos , Autoantígenos/química , Células HEK293 , Humanos , Naftiridinas/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Ribonucleoproteínas/química , Serina/metabolismo , Sirolimo/farmacologia , Treonina/metabolismo , Tirosina/metabolismo
19.
Biofactors ; 47(1): 126-134, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33469985

RESUMO

Intraneuronal accumulation of hyperphosphorylated tau is a pathological hallmark of several neurodegenerative disorders, including Alzheimer's disease. Phosphorylation plays a crucial role in modulating the tau-microtubule interaction and the ability of the protein to aggregate, but despite efforts during the past decades, the real identity of the kynases involved in vivo remains uncertain. Here, for the first time, we demonstrate that the cGMP-dependent protein kinase G (PKG) phosphorylates tau in both in vitro and in vivo models. More intriguingly, we provide evidence that PKG phosphorylates tau at Ser214 but not at Ser202, a condition that could reduce the pathological aggregation of the protein shifting tau from a pro-aggregant to a neuroprotective anti-aggregant conformation.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas tau/metabolismo , Animais , Células Cultivadas , GMP Cíclico/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Agregados Proteicos , Ratos Sprague-Dawley , Serina/metabolismo , Treonina/metabolismo , Proteínas tau/química
20.
Food Chem ; 341(Pt 1): 128066, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33035859

RESUMO

An important relationship exists between the changes in glycation sites and structure of proteins. A combination of liquid chromatography and tandem mass spectrometry was used to identify the glycation information from ovalbumin (OVA) after dextran (Dex) conjugation under water heating (WH) or microwave heating (MH) conditions. After Dex conjugation, 12O-linked glycation sites were identified in the OVA-Dex-MH conjugates, whereas 6O-linked, 2N-linked glycation sites were detected in the OVA-Dex-WH conjugates. These findings indicate that the amino acids at different positions in OVA molecular structure have different glycation reactivity under MH or WH induction systems. In addition, ß-sheet and ß-turn structures showed high glycation reactivity. The increased surface hydrophobicity of OVA-Dex conjugates was possibly attributed to the glycation sites that were mainly found in hydrophilic amino acids. Our study provides useful information for the glycation mechanism research of OVA and Dex.


Assuntos
Dextranos/metabolismo , Ovalbumina/química , Ovalbumina/metabolismo , Cromatografia Líquida , Dextranos/química , Produtos Finais de Glicação Avançada , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Micro-Ondas , Serina/metabolismo , Espectrometria de Massas em Tandem , Treonina/metabolismo
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