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1.
Nat Commun ; 10(1): 3099, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308373

RESUMO

The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Engenharia Metabólica/métodos , Optogenética/métodos , Fitocromo/genética , Proteínas Quinases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Luz , Ficobilinas/biossíntese , Ficocianina/biossíntese , Fitocromo/metabolismo , Regiões Promotoras Genéticas/efeitos da radiação , Proteínas Quinases/metabolismo
2.
Comput Biol Chem ; 80: 480-497, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31174160

RESUMO

Laminin-111 is a trimeric glycoprotein of the extracellular matrix (ECM) that holds a significant role in cell adhesion, migration and differentiation. Laminin-111 is the most studied laminin isoform, composed of three chains; α1, ß1 and γ1. Phosphorylation is the most common eukaryotic post - translational modification and has regulatory effect on protein function. Using bioinformatic tools we computationally predicted all the possible phosphorylation sites on human laminin α1-chain sequence (LAMA1) according to kinases binding motifs. Thus, we predicted, for the first time, the possibly responsible kinases for fifteen of the nineteen already published experimentally observed phosphorylated residues in LAMA1. Searching the literature extensively, we recorded all the known functional sites (active sites) in LAMA1. We combined the experimentally observed and predicted phosphorylated residues as well as the active sites in LAMA1, generating an analytic phosphorylation map of human laminin α1-chain, which is useful for further analysis. Our results indicated fourteen kinases that might be important for the phosphorylation of human laminin α1-chain, out of which three kinases with reported ecto-phosphorylation activity (PKA, PKC and CKII) were suggested to have a more significant role. Six cancer associated-active sites were correlated with kinases, three out which were correlated with only the above ecto - kinases.


Assuntos
Laminina/química , Laminina/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional
3.
Curr Top Med Chem ; 19(6): 467-485, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31184298

RESUMO

BACKGROUND: Kinases are key modulators in regulating diverse range of cellular activities and are an essential part of the protein-protein interactome. Understanding the interaction of kinases with different substrates and other proteins is vital to decode the cell signaling machinery as well as causative mechanism for disease onset and progression. OBJECTIVE: The objective of this review is to present all studies on the structure and function of few important kinases and highlight the protein-protein interaction (PPI) mechanism of kinases and the kinase specific interactome databases and how such studies could be utilized to develop anticancer drugs. METHODS: The article is a review of the detailed description of the various domains in kinases that are involved in protein-protein interactions and specific inhibitors developed targeting these PPI domains. RESULTS: The review has surfaced in depth the interacting domains in key kinases and their features and the roles of PPI in the human kinome and the various signaling cascades that are involved in certain types of cancer. CONCLUSION: The insight availed into the mechanism of existing peptide inhibitors and peptidomimetics against kinases will pave way for the design and generation of domain specific peptide inhibitors with better productivity and efficiency and the various software and servers available can be of great use for the identification and analysis of protein-protein interactions.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Antineoplásicos/química , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Peptidomiméticos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/química
4.
BMC Plant Biol ; 19(1): 256, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196007

RESUMO

BACKGROUND: Appropriate brassinosteroid (BR) signal strength caused by exogenous application or endogenous regulation of BR-related genes can increase crop yield. However, precise control of BR signals is difficult and can cause unstable effects and failure to reach full potential. Phosphorylated BRASSINOSTEROID INSENSITIVE1 (BRI1), the rate-limiting receptor in BR signalling, transduces BR signals, and we recently demonstrated that modifying BRI1 phosphorylation sites alters BR signal strength and botanical characteristics in Arabidopsis. However, the functions of such phosphorylation sites in agronomic characteristics of crops remain unclear. RESULTS: In this work, we investigated the roles of tomato SlBRI1 threonine-1050 (Thr-1050). SlBRI1 mutant cu3-abs1 plants expressing SlBRI1 with a non-phosphorylatable Thr-1050 (T1050A), with a wild-type SlBRI1 transformant used as a control, were examined. The results showed enhanced autophosphorylation of SlBRI1 and BR signal strength for cu3-abs1 harbouring T1050A, which promoted yield through increased plant expansion, leaf area, fruit weight and fruit number per cluster but reduced nutrient contents, including ascorbic acid and soluble sugar levels. Moreover, plant height, stem diameter, and internodal distance were similar between the transgenic plants. CONCLUSION: Our results reveal the biological role of Thr-1050 in tomato and provide a molecular basis for establishing high-yield crops by precisely controlling BR signal strength via phosphorylation site modification.


Assuntos
Brassinosteroides/metabolismo , Frutas/crescimento & desenvolvimento , Lycopersicon esculentum/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Proteínas Quinases/fisiologia , Transdução de Sinais , Lycopersicon esculentum/genética , Mutação , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo
5.
BMC Plant Biol ; 19(1): 190, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068146

RESUMO

BACKGROUND: The functional characteristics of SLAC/SLAH family members isolated from Arabidopsis thaliana, poplar, barley and rice have been comprehensively investigated. However, there are no reports regarding SLAC/SLAH family genes from Rosaceae plants. RESULTS: In this study, the function of PbrSLAH3, which is predominately expressed in pear (Pyrus bretschneideri) root, was investigated. PbrSLAH3 can rescue the ammonium toxicity phenomenon of slah3 mutant plants under high-ammonium/low-nitrate conditions. In addition, yeast two-hybrid and bimolecular fluorescence complementation assays confirmed that PbrSLAH3 interacts with PbrCPK32. Moreover, when PbrSLAH3 was co-expressed with either the Arabidopsis calcium-dependent protein kinase (CPK) 21 or PbrCPK32 in Xenopus oocytes, yellow fluorescence was emitted from the oocytes and typical anion currents were recorded in the presence of extracellular NO3-. However, when PbrSLAH3 alone was injected, no yellow fluorescence or anion currents were recorded, suggesting that anion channel PbrSLAH3 activity was controlled through phosphorylation. Finally, electrophysiological and transgene results showed that PbrSLAH3 was more permeable to NO3- than Cl-. CONCLUSION: We suggest that PbrSLAH3 crossing-talk with PbrCPK32 probably participate in transporting of nitrate nutrition in pear root.


Assuntos
Canais Iônicos/metabolismo , Nitratos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Pyrus/enzimologia , Compostos de Amônio/toxicidade , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Mutação/genética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pyrus/efeitos dos fármacos , Pyrus/genética , Xenopus
6.
mSphere ; 4(3)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043517

RESUMO

The early divergent protozoan parasite Trypanosoma brucei alternates between the insect vector and the mammalian hosts during its life cycle and proliferates through binary cell fission. The cell cycle control system in T. brucei differs substantially from that in its mammalian hosts and possesses distinct mitosis-cytokinesis checkpoint controls between two life cycle stages, the procyclic form and the bloodstream form. T. brucei undergoes an unusual mode of cytokinesis, which is controlled by a novel signaling cascade consisting of evolutionarily conserved protein kinases and trypanosome-specific regulatory proteins in the procyclic form. However, given the distinct mitosis-cytokinesis checkpoints between the two forms, it is unclear whether the cytokinesis regulatory pathway discovered in the procyclic form also operates in a similar manner in the bloodstream form. Here, we showed that the three regulators of cytokinesis initiation, cytokinesis initiation factor 1 (CIF1), CIF2, and CIF3, are interdependent for subcellular localization but not for protein stability as in the procyclic form. Further, we demonstrated that KLIF, a regulator of cytokinesis completion in the procyclic form, plays limited roles in cytokinesis in the bloodstream form. Finally, we showed that the cleavage furrow-localizing protein FRW1 is required for cytokinesis initiation in the bloodstream form but is nonessential for cytokinesis in the procyclic form. Together, these results identify conserved and life cycle-specific functions of cytokinesis regulators, highlighting the distinction in the regulation of cytokinesis between different life cycle stages of T. brucei IMPORTANCE The early divergent protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. This parasite has a complex life cycle by alternating between the insect vector and the mammalian hosts and proliferates by binary cell fission. The control of cell division in trypanosomes appears to be distinct from that in its human host and differs substantially between two life cycle stages, the procyclic (insect) form and the bloodstream form. Cytokinesis, the final step of binary cell fission, is regulated by a novel signaling cascade consisting of two evolutionarily conserved protein kinases and a cohort of trypanosome-specific regulators in the procyclic form, but whether this signaling pathway operates in a similar manner in the bloodstream form is unclear. In this report, we performed a functional analysis of multiple cytokinesis regulators and discovered their distinct functions and regulations in the bloodstream form.


Assuntos
Citocinese , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Bovina/sangue , Animais , Bovinos , Regulação da Expressão Gênica , Estágios do Ciclo de Vida , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo
7.
Plant Physiol Biochem ; 139: 660-671, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31048123

RESUMO

In Arabidopsis, the serine/threonine protein kinase Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 2 polypeptide (EIN2) functions are key negative and positive components, respectively, in the ethylene signalling route. Here, we report on an in silico study of members of the CTR1-like and EIN2-like polypeptide families from poplars. The expression of CTR1-like and EIN2-like genes such as Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a was studied in Populus tremula buds and leaves in response to dehydration, various light conditions and under senescence. In buds under dehydration, the maximal fold-change of the Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a expression level recorded almost identical values. This suggests that maintenance of a constant ratio between the transcript levels of genes encoding positive and negative ethylene signalling components is required under stress. The expression of the studied genes was 1.4-to 3-fold higher in response to darkness, but 4.5- to 51.2-fold and 21.6- to 51.2-fold higher under the early and moderate leaf senescence, respectively. It is worth noting that the senescence-related Ptre-EIN2a and Ptre-CTR3a expression profiles were very similar. Using in vitro assays, we demonstrated the ability of the catalytic domain of Ptre-CTR1 to phosphorylate the Ptre-EIN2a-like polypeptide, which is similar to that in Arabidopsis. The target substrate, the Ptre-CEND2a polypeptide (C-terminal part of Ptre-EIN2a), was only phosphorylated by the protein kinase Ptre-CTR1 and not by Ptre-CTR3. Moreover, the addition of Ptre-CTR3 polypeptides (-CTR3a or -CTR3b forms) to the reaction mixture had an inhibitory effect on Ptre-CTR1 auto- and trans-phosphorylation. In contrast to Ptre-CTR1, Ptre-CTR3 may act as a positive regulator in ethylene signalling in poplar; however, this hypothesis requires in vivo confirmation. Thus, the ethylene signalling route in poplar seems to be under the control of certain additional mechanisms which have not been reported in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Populus/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Etilenos/metabolismo , Fosforilação , Folhas de Planta/metabolismo , Populus/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
8.
Molecules ; 24(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086079

RESUMO

As calcium signal sensors, calcium-dependent protein kinases (CPKs) play vital roles in stimulating the production of secondary metabolites to participate in plant development and response to environmental stress. However, investigations of the Glycyrrhiza uralensis CPK family genes and their multiple functions are rarely reported. In this study, a total of 23 GuCPK genes in G. uralensis were identified, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, and promoter cis-acting elements were analyzed. Ten GuCPKs showed root-specific preferential expressions, and GuCPKs indicated different expression patterns under treatments of CaCl2 and NaCl. In addition, under 2.5 mM of CaCl2 and 30 mM of NaCl treatments, the diverse, induced expression of GuCPKs and significant accumulations of glycyrrhizic acid and flavonoids suggested the possible important function of GuCPKs in regulating the production of glycyrrhizic acid and flavonoids. Our results provide a genome-wide characterization of CPK family genes in G. uralensis, and serve as a foundation for understanding the potential function and regulatory mechanism of GuCPKs in promoting the biosynthesis of glycyrrhizic acid and flavonoids under salt stress.


Assuntos
Flavonoides/metabolismo , Glycyrrhiza uralensis/efeitos dos fármacos , Glycyrrhiza uralensis/metabolismo , Ácido Glicirrízico/metabolismo , Proteínas Quinases/metabolismo , Cloreto de Cálcio/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glycyrrhiza uralensis/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Quinases/genética , Estresse Salino , Cloreto de Sódio/farmacologia
9.
Environ Toxicol ; 34(8): 902-911, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31044527

RESUMO

Osteosarcoma (OS) is a tumor entity that can cause a large number of cancer-related deaths. Although chemotherapy can decrease proliferation and increase apoptosis of human OS cells, the clinical prognosis remains poor. Fisetin is a flavonol found in fruits and vegetables and is reported to inhibit cell growth in numerous cancers. But the molecular mechanism underlying fisetin in human OS cells is not clear. It is known that sterile-alpha motif and leucine zipper containing kinase (ZAK), a kinase in the MAP3K family, is involved in various cell processes, including proliferation and apoptosis. In our lab, we have demonstrated that overexpression of ZAK can induce apoptosis in human OS cells. In the previous studies, MAP4K, the upstream of MAP3K, can act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway. Turning on the Hippo pathway can decrease proliferation and otherwise cause cell apoptosis in cancer cells. In this study, we found that fisetin can upregulate ZAK expression to induce the Hippo pathway and mediate the activation of JNK/ERK, the downstream of ZAK, to trigger cell apoptosis via AP-1 dependent manner in human OS cells. These findings reveal a novel molecular mechanism underlying fisetin effect on human OS cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases , Osteossarcoma/metabolismo , Proteínas Quinases/metabolismo , Apoptose , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Osteossarcoma/enzimologia , Osteossarcoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Proteínas Supressoras de Tumor/metabolismo
10.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075880

RESUMO

The interaction between programmed cell death protein (PD-1) and its ligand (PD-L1) is one of the main pathways used by some tumors to escape the immune response. In recent years, immunotherapies based on the use of antibodies against PD-1/PD-L1 have been postulated as a great promise for cancer treatment, increasing total survival compared to standard therapy in different tumors. Despite the hopefulness of these results, a significant percentage of patients do not respond to such therapy or will end up evolving toward a progressive disease. Besides their role in PD-L1 expression, altered protein kinases in tumor cells can limit the effectiveness of PD-1/PD-L1 blocking therapies at different levels. In this review, we describe the role of kinases that appear most frequently altered in tumor cells and that can be an impediment for the success of immunotherapies as well as the potential utility of protein kinase inhibitors to enhance the response to such treatments.


Assuntos
Antígeno B7-H1/metabolismo , Imunoterapia , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Quinases/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos
11.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083521

RESUMO

ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) act as key regulators of vesicle trafficking in all eukaryotes. In Arabidopsis, there are eight ARF-GEFs, including three members of the GBF1 subfamily and five members of the BIG subfamily. These ARF-GEFs have different subcellular localizations and regulate different trafficking pathways. Until now, the roles of these BIG-subfamily ARF-GEFs have not been fully revealed. Here, analysis of the BIGs expression patterns showed that BIG3 and BIG5 have similar expression patterns. big5-1 displayed a dwarf growth and big3-1 big5-1 double mutant showed more severe defects, indicating functional redundancy between BIG3 and BIG5. Moreover, both big5-1 and big3-1 big5-1 exhibited a reduced sensitivity to Brassinosteroid (BR) treatment. Brefeldin A (BFA)-induced BR receptor Brassinosteroid insensitive 1 (BRI1) aggregation was reduced in big5-1 mutant, indicating that the action of BIG5 is required for BRI1 recycling. Furthermore, BR-induced dephosphorylation of transcription factor BZR1 was decreased in big3-1 big5-1 double mutants. The introduction of the gain-of-function of BZR1 mutant BZR1-1D in big3-1 big5-1 mutants can partially rescue the big3-1 big5-1 growth defects. Our findings revealed that BIG5 functions redundantly with BIG3 in plant growth and gravitropism, and BIG5 participates in BR signal transduction pathway through regulating BRI1 trafficking.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Gravitropismo , Desenvolvimento Vegetal , Proteínas Quinases/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/farmacologia , Teste de Complementação Genética , Gravitropismo/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Inflorescência/efeitos dos fármacos , Inflorescência/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Desenvolvimento Vegetal/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
12.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091743

RESUMO

As one of the typical Maillard reaction products, furosine has been widely reported in a variety of heat-processed food. Though furosine was shown to be toxic on organs, its toxicity mechanism is still unclear. The present study aimed to investigate the toxicity mechanism of furosine in liver tissue. An intragastric gavage mice model (42-day administration, 0.1/0.25/0.5 g/kg of furosine per day) and a mice primary hepatocyte model were employed to investigate the toxicity mechanism of furosine on mice liver tissue. A metabonomics analysis of mice liver, serum, and red blood cells (RBC) was performed. The special metabolic mediator of furosine, lysophosphatidylcholine 18:0 (LPC (18:0)) was identified. Then, the effect of the upstream gene phospholipase A2 gamma (PLA2-3) on LPC (18:0), as well as the effect of furosine (100 mg/L) on the receptor-interacting serine/threonine-protein kinase (RIPK)1/RIPK3/mixed lineage kinase domain-like protein (MLKL) pathway and inflammatory factors, was determined in liver tissue and primary hepatocytes. PLA2-3 was found to regulate the level of LPC (18:0) and activate the expression of RIPK1, RIPK3, P-MLKL, and of the inflammatory factors including tumor necrosis factor α (TNF-α) and interleukin (IL-1ß), both in liver tissue and in primary hepatocytes. Upon treatment with furosine, the upstream sensor PLA2-3 activated the RIPK1/RIPK3/MLKL necroptosis pathway and caused inflammation by regulating the expression of LPC (18:0), which further caused liver damage.


Assuntos
Produtos Finais de Glicação Avançada/toxicidade , Hepatócitos/metabolismo , Lisina/análogos & derivados , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Morte Celular , Células Cultivadas , Feminino , Produtos Finais de Glicação Avançada/metabolismo , Interleucina-1beta/metabolismo , Lisina/metabolismo , Lisina/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator de Necrose Tumoral alfa/metabolismo
13.
Plant Cell Physiol ; 60(8): 1804-1810, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31119298

RESUMO

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Quitina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fosforilação/genética , Fosforilação/fisiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
14.
Comput Biol Chem ; 80: 324-332, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31078911

RESUMO

Various protein kinases are implicated in the pathogenesis of human cervical cancer and many kinase inhibitors have been used to regulate the activity of protein kinases involved in the disease signaling networks. In the present study, a systematic kinase-inhibitor interactome is created for various small-molecule inhibitors across diverse cervical cancer-related kinases by using ontology enrichment, molecular docking, dynamics simulation and energetics analysis. The interactome profile is examined in detail with heatmap analysis and heuristic clustering to derive promising inhibitors that are highly potential to target the kinome of human cervical cancer in a multi-target manner. A number of hit and unhit inhibitors are selected and their cell-suppressing effects are tested against human cervical carcinoma HeLa, from which several inhibitor compounds with high cytotoxicity are successfully identified. A further kinase assay confirms that these inhibitors can generally target their noncognate kinases HER3 and BRaf in cervical cancer with a high or moderate activity; the activity profile are comparable with or even better than that of cognate kinases inhibitors, with IC50 values ranging between 4.8 and 340.6 nM for HER3 and between 37.2 and 638.2 nM for BRaf. This work would help to identify those unexpected kinase-inhibitor interactions in human cervical cancer and to develop new and efficient therapeutic strategy combating the disease.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Neoplasias do Colo do Útero/enzimologia , Domínio Catalítico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Ontologia Genética , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética
15.
Cell Mol Life Sci ; 76(14): 2789-2797, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31037337

RESUMO

The heterotrimeric carboxy-terminal domain kinase I (CTDK-I) in yeast is a cyclin-dependent kinase complex that is evolutionally conserved throughout eukaryotes and phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (RNApII) on the second-position serine (Ser2) residue of YSPTSPS heptapeptide repeats. CTDK-I plays indispensable roles in transcription elongation and transcription-coupled processing, such as the 3'-end processing of nascent mRNA transcripts. However, recent studies have revealed additional roles of CTDK-I beyond its primary effect on transcription by RNApII. Here, we describe recent advances in the regulation of genomic stability and rDNA integrity by CTDK-I and highlight the previously underappreciated cellular roles of CTDK-I in rRNA synthesis by RNA polymerase I and translational initiation and elongation. These multiple roles of CTDK-I throughout the cell expand our understanding of how this complex functions to coordinate diverse cellular processes through gene expression and how the human orthologue exerts its roles in diseased states such as tumorigenesis.


Assuntos
Biossíntese de Proteínas , Proteínas Quinases/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/metabolismo , Animais , Humanos
16.
Croat Med J ; 60(2): 121-126, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31044583

RESUMO

Due to very limited therapeutic options, ischemic brain injury is one of the leading causes of death and lifelong disability worldwide, which imposes enormous public health burden. One of the main events occurring with ischemic brain stroke is cell death. Necroptosis is a type of cell death described as a regulated necrosis characterized by cell membrane disruption mediated by phosphorylated mixed lineage kinase like protein (MLKL). It can be triggered by activation of death receptors (eg, FAS, TNFR1), which lead to receptor-interacting serine/threonine-protein kinase 3 (RIPK3) activation by RIPK1 in the absence of active caspase-8. Here, we review articles that have reported that necroptosis significantly contributes to negative events occurring with the ischemic brain stroke, and that its inhibition is protective both in vitro and in vivo. We also review articles describing positive effects obtained by reducing necroptosis, including the reduction of infarct volume and improved functional recovery in animal models. Since necroptosis is characterized by cell content leakage and subsequent inflammation, in addition to reducing cell death, inhibition of necroptosis in ischemic brain stroke also reduces some inflammatory cytokines. By comparing various approaches in inhibition of necroptosis, we analyze the achieved effects from the perspective of controlling necroptosis as a part of future therapeutic interventions in brain ischemia.


Assuntos
Isquemia Encefálica/fisiopatologia , Morte Celular , Inibidores Enzimáticos/uso terapêutico , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Inibidores Enzimáticos/farmacologia , Humanos , Inflamação/metabolismo , Necrose , Fosforilação , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral , Acidente Vascular Cerebral
17.
BMC Plant Biol ; 19(1): 202, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096905

RESUMO

BACKGROUND: The Fertilization-related kinases (FRK) form a class that belongs to the MEKK subfamily of plant MAPKKKs. It was recently shown that FRK class kinases expanded during angiosperm evolution, reaching their maximum numbers in the lineage leading to solanaceous species and culminating in the Solanum genus where they account for more than 40% of the total MEKKs. The first members studied, ScFRK1 and ScFRK2 were shown to play a pivotal role in gametophyte development in the wild potato species Solanum chacoense. RESULTS: ScFRK3 is also involved in gametophyte development. ScFRK3 is expressed in developing pollen and young ovules, reaching its highest level immediately after meiosis and during the mitosis steps in both gametophytes. Hence, three independent lines of ScFRK3 RNAi mutant plants showed decreased number of seeds per fruit. We also observed an important number of degenerated embryo sac in mature ovary. Analysis of ovule development showed that most embryo sac did not enter mitosis I in ScFRK3 RNAi mutant plants. Severe lethality was also observed during male gametophyte development, pollen being arrested before mitosis I, as observed in the female gametophyte. Obvious defects in vegetative organs were not observed, emphasizing the reproductive roles of the FRK class kinases. To isolate MAP kinases acting downstream of ScFRK3, a de novo S. chacoense transcriptome from male and female reproductive organs was assembled. Of the five ScMKKs and 16 ScMPKs retrieved, only the ScMKK3 interacted with ScFRK3, while only the ScMPK13 interacted with ScMKK3, leading to an apparent single three-tiered canonical MAP kinase cascade combination involving ScFRK3-ScMKK3-ScMPK13. CONCLUSIONS: The ScFRK3 MAPKKK is involved in a signaling cascade that regulates both male and female gamete development, and most probably act upstream of ScMKK3 and ScMPK13.


Assuntos
Óvulo Vegetal/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Pólen/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Solanum/crescimento & desenvolvimento , Hibridização In Situ , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , RNA de Plantas/metabolismo , Solanum/enzimologia , Solanum/genética , Técnicas do Sistema de Duplo-Híbrido
18.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035605

RESUMO

Dual specificity phosphatases (DUSPs) have a well-known role as regulators of the immune response through the modulation of mitogen-activated protein kinases (MAPKs). Yet the precise interplay between the various members of the DUSP family with protein kinases is not well understood. Recent multi-omics studies characterizing the transcriptomes and proteomes of immune cells have provided snapshots of molecular mechanisms underlying innate immune response in unprecedented detail. In this study, we focus on deciphering the interplay between members of the DUSP family with protein kinases in immune cells using publicly available omics datasets. Our analysis resulted in the identification of potential DUSP-mediated hub proteins including MAPK7, MAPK8, AURKA, and IGF1R. Furthermore, we analyzed the association of DUSP expression with TLR4 signaling and identified VEGF, FGFR, and SCF-KIT pathway modules to be regulated by the activation of TLR4 signaling. Finally, we identified several important kinases including LRRK2, MAPK8, and cyclin-dependent kinases as potential DUSP-mediated hubs in TLR4 signaling. The findings from this study have the potential to aid in the understanding of DUSP signaling in the context of innate immunity. Further, this will promote the development of therapeutic modalities for disorders with aberrant DUSP signaling.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Imunomodulação , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Evolução Biológica , Células Sanguíneas/metabolismo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteoma , Proteômica/métodos
19.
Gene ; 709: 75-83, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129249

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative pathogen for porcine reproductive and respiratory syndrome (PRRS), which lead to huge loss to porcine industry. RACK1 (receptor of activated protein C kinase 1) was first identified as a receptor for protein kinase C. Mounting evidence demonstrated that RACK1 played diverse roles in NF-κB activation and virus infections. We previously reported that siRNA knockdown of RACK1 inhibited PRRSV replication in Marc-145 cells, abrogated NF-κB activation induced by PRRSV infection and reduced the viral titer. Here we established a Marc-145 cell line which could stably overexpress RACK1 to consolidate our findings. Based on the data from RT-qPCR, western blot, immunofluorescence staining, cytopathic effects and viral titer analysis, we concluded that overexpression of RACK1 could enhance the replication of PRRSV in Marc-145 cells and promote the NF-κB activation via upregulating TRAF2 expression and its phosphorylation. Marc-145 cells overexpressing RACK1exhibited severe cytopathic effects post infection with PRRSV and elevated the viral titer. Taken together, RACK1 plays an essential role for PRRSV replication in Marc-145 cells and NF-κB activation. The results presented here shed more light on the understanding of the molecular mechanisms underlying PRRSV infection and its subsequent NF-κB activation. Therefore, we anticipate RACK1 as a promising target for PRRS control.


Assuntos
NF-kappa B/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Receptores de Quinase C Ativada/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Replicação Viral/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Macaca mulatta , Fosforilação , Proteínas Quinases/metabolismo , Receptores de Quinase C Ativada/metabolismo , Transdução de Sinais/genética , Ativação Transcricional , Regulação para Cima
20.
J Agric Food Chem ; 67(24): 6809-6818, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31134808

RESUMO

Strategies to increase feed intake are of great importance for producing more meat in swine production. Intestinal and hypothalamic amino acid receptors are found to largely participate in feed intake regulation. The purpose of the current research is to study the function of branched-chain amino acid (BCAA) supplementation in the regulation of feed intake through sensors that can detect amino acids in piglets. Twenty-four piglets were assigned one of four treatments and fed one of the experimental diets for either a short period (Expt. 1) or a long period (Expt. 2): a normal protein diet (NP, 20.04% CP), a reduced-protein diet (RP, 17.05% CP), or a reduced-protein test diet supplemented with one of two doses of BCAAs (BCAA1, supplemented with 0.13% l-isoleucine, 0.09% l-leucine, and 0.23% l-valine; BCAA2, supplemented with the 150% standardized ileal digestibility BCAA requirement, as recommended by the National Research Council (2012)). In Expt. 1, no differences were observed in the feed intake among piglets fed different diets ( P > 0.05). In Expt. 2, compared with the RP group, the feed intake of piglets was significantly increased after sufficient BCAAs were supplemented in the BCAA1 group, which was associated with decreased cholecystokinin secretion ( P < 0.05), down-regulated expression of type-1 taste receptors 1/3 (T1R1/T1R3) in the intestine, as well as increased expression of pro-opiomelanocortin, activated general control nonderepressible 2 (GCN2), and eukaryotic initiation factor 2α (eIF2α) in the hypothalamus ( P < 0.05). However, the feed intake was decreased for unknown reasons when the piglets were fed a BCAA over-supplemented diet. Our study confirmed that a BCAA-deficient diet inhibited feed intake through two potential ways: regulating the amino acid T1R1/T1R3 receptor in the intestine or activating GCN2/eIF2α pathways in the hypothalamus.


Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Ingestão de Alimentos , Hipotálamo/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Aminoácido/metabolismo , Suínos/fisiologia , Ração Animal/análise , Animais , Colecistocinina/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Comportamento Alimentar , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Suínos/genética
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