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1.
Gene ; 723: 144134, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589960

RESUMO

Viral kinases are known to undergo autophosphorylation and also phosphorylate viral and host substrates. Viral kinases have been implicated in various diseases and are also known to acquire host kinases for mimicking cellular functions and exhibit virulence. Although substantial analyses have been reported in the literature on diversity of viral kinases, there is a gap in the understanding of sequence and structural similarity among kinases from different classes of viruses. In this study, we performed a comprehensive analysis of protein kinases encoded in viral genomes. Homology search methods have been used to identify kinases from 104,282 viral genomic datasets. Serine/threonine and tyrosine kinases are identified only in 390 viral genomes. Out of seven viral classes that are based on nature of genetic material, only viruses having double-stranded DNA and single-stranded RNA retroviruses are found to encode kinases. The 716 identified protein kinases are classified into 63 subfamilies based on their sequence similarity within each cluster, and sequence signatures have been identified for each subfamily. 11 clusters are well represented with at least 10 members in each of these clusters. Kinases from dsDNA viruses, Phycodnaviridae which infect green algae and Herpesvirales that infect vertebrates including human, form a major group. From our analysis, it has been observed that the protein kinases in viruses belonging to same taxonomic lineages form discrete clusters and the kinases encoded in alphaherpesvirus form host-specific clusters. A comprehensive sequence and structure-based analysis enabled us to identify the conserved residues or motifs in kinase catalytic domain regions across all viral kinases. Conserved sequence regions that are specific to a particular viral kinase cluster and the kinases that show close similarity to eukaryotic kinases were identified by using sequence and three-dimensional structural regions of eukaryotic kinases as reference. The regions specific to each viral kinase cluster can be used as signatures in the future in classifying uncharacterized viral kinases. We note that kinases from giant viruses Marseilleviridae have close similarity to viral oncogenes in the functional regions and in putative substrate binding regions indicating their possible role in cancer.


Assuntos
Proteínas Quinases/química , Proteínas Quinases/genética , Vírus/classificação , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Variação Genética , Fosforilação , Filogenia , Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Vírus/enzimologia , Vírus/patogenicidade
2.
Nature ; 571(7766): 565-569, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31316206

RESUMO

Parkinson's disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles1,2. An early-onset form of Parkinson's disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes3. PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells4, but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo5-8. It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens9, which suggests that autoimmune mechanisms participate in the aetiology of Parkinson's disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1-/- mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with L-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson's disease, which highlights the relevance of the gut-brain axis in the disease10.


Assuntos
Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/fisiopatologia , Intestinos/microbiologia , Doença de Parkinson/genética , Doença de Parkinson/microbiologia , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Animais , Apresentação do Antígeno/imunologia , Autoantígenos/imunologia , Axônios/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Modelos Animais de Doenças , Neurônios Dopaminérgicos/imunologia , Neurônios Dopaminérgicos/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/patologia , Feminino , Intestinos/imunologia , Intestinos/patologia , Levodopa/uso terapêutico , Masculino , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/patologia , Neostriado/imunologia , Neostriado/microbiologia , Neostriado/patologia , Neostriado/fisiopatologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/fisiopatologia , Proteínas Quinases/imunologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
3.
Cell Mol Life Sci ; 76(19): 3827-3841, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302748

RESUMO

The Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine-threonine kinases that have been implicated in DNA replication, DNA repair, transcription, chromatin structure, viral latency, cell cycle checkpoint control and chromosomal stability in various organisms. The functions of the TLKs appear to depend largely on their ability to regulate the H3/H4 histone chaperone ASF1, although numerous TLK substrates have been proposed. Over the last few years, a clearer picture of TLK function has emerged through the identification of new partners, the definition of specific roles in development and the elucidation of their structural and biochemical properties. In addition, the TLKs have been clearly linked to human disease; both TLK1 and TLK2 are frequently amplified in human cancers and TLK2 mutations have been identified in patients with neurodevelopmental disorders characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. A better understanding of the substrates, regulation and diverse roles of the TLKs is needed to understand their functions in neurodevelopment and determine if they are viable targets for cancer therapy. In this review, we will summarize current knowledge of TLK biology and its potential implications in development and disease.


Assuntos
Instabilidade Genômica , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Epigênese Genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transtornos do Neurodesenvolvimento/genética , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia
4.
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
5.
PLoS Genet ; 15(6): e1008206, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194741

RESUMO

The septation initiation network (SIN), composed of a conserved SepH (Cdc7p) kinase cascade, plays an essential role in fungal cytokinesis/septation and conidiation for asexual reproduction, while the mitogen-activated protein kinase (MAPK) pathway depends on successive signaling cascade phosphorylation to sense and respond to stress and environmental factors. In this study, a SepH suppressor-PomA in the filamentous fungus A. nidulans is identified as a negative regulator of septation and conidiation such that the pomA mutant is able to cure defects of sepH1 in septation and conidiation and overexpression of pomA remarkably suppresses septation. Under the normal cultural condition, SepH positively regulates the phosphorylation of MAPK-HogA, while PomA reversely affects this process. In the absence of PbsB (MAPKK, a putative upstream member of HogA), PomA and SepH are unable to affect the phosphorylation level of HogA. Under the osmostress condition, the induced phosphorylated HogA is capable of bypassing the requirement of SepH, a key player for early events during cytokinesis but not for MobA/SidB, the last one in the core SIN protein kinase cascade, indicating the osmotic stimuli-induced septation is capable of bypassing requirement of SepH but unable to bypass the whole SIN requirement. Findings demonstrate that crosstalk exists between the SIN and MAPK pathways. PomA and SepH indirectly regulate HogA phosphorylation through affecting HogA-P upstream kinases.


Assuntos
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Reprodução Assexuada/genética , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Citocinese/genética , Mutação/genética , Proteínas Nucleares/genética , Pressão Osmótica , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/genética
6.
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
7.
Nat Protoc ; 14(6): 1863-1883, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076662

RESUMO

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/efeitos da radiação , Engenharia de Proteínas/métodos , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/efeitos da radiação , Animais , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/efeitos da radiação , Linhagem Celular , Clonagem Molecular/métodos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Luz , Camundongos , Modelos Moleculares , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sirolimo/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
8.
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
9.
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
10.
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
11.
Yi Chuan ; 41(5): 430-438, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31106779

RESUMO

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Quinases/genética , Flores/genética , Regulação da Expressão Gênica de Plantas
12.
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
13.
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
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.
Plant Dis ; 103(7): 1450-1457, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31107641

RESUMO

Northern corn leaf blight (NCLB) caused by Exserohilum turcicum is the most common and economically significant fungal leaf disease of corn in Ontario, Canada. During the past 10 years in Ontario, severity and incidence of NCLB have increased, possibly owing to the appearance of new races. Several races have been identified in various parts of the world; however, information on occurrence and distribution of races in Ontario is lacking. In the current study, 677 single conidial isolates of E. turcicum were isolated from 687 symptomatic leaf samples collected between 2012 and 2016. These isolates were evaluated for pathogenicity on six corn differential inbreds (A619, A619Ht1, A619Ht2, A619Ht3, A632Htn1, and H102Htm1) under controlled environmental conditions and then grouped into 17 physiological races (0, 1, 2, 3, M, N, 12, 1M, 1N, 3M, 13M, 12N, 13N, 1MN, 12MN, 13MN, 123MN) based on the reaction of the inbreds to infection (resistant or susceptible). Four races (0, 1M, 1N, and 1MN) were most frequent, with an isolation frequency of 13, 10, 12, and 41%, respectively. Seventy-six percent of the isolates were virulent on more than one Ht resistance gene, with 2.4% (16 isolates) virulent on all five Ht resistance genes used in this study. Further analysis of the distribution of races in four regions over the years revealed that the occurrence and distribution of the races changed with time in Ontario. Overall, the frequency of virulence of the 677 isolates screened on the differentials with resistance genes Ht1, Ht2, Ht3, Htm1, and Htn1 varied from 6 to 81% (Ht1 81%, Ht2 6%, Ht3 12%, Htm1 64%, and Htn1 64%). Virulent isolates produced fewer lesions on the Htm1 differential, and smaller lesions that were slower and having less sporulation on the Htn1 differential, compared with infection of the differentials with Ht1, Ht2, and Ht3 resistance genes. Virulence frequency also changed within the four geographical regions of Ontario, with fewer isolates virulent on all resistance genes in eastern Ontario compared with southern and western Ontario. Isolates from southern Ontario had greater virulence frequency against Ht1 and Htm1, whereas isolates from western Ontario were more frequently virulent on Ht1 and Htn1. The information generated in this study on the distribution of E. turcicum races in Ontario corn will help growers to select appropriate hybrids with required resistance genes and will assist seed companies in deploying resistance genes in corn hybrids across the province or within a particular region.


Assuntos
Ascomicetos/classificação , Ascomicetos/patogenicidade , Zea mays/microbiologia , Ascomicetos/enzimologia , Genes Fúngicos/genética , Especificidade de Hospedeiro , Ontário , Doenças das Plantas/microbiologia , Proteínas Quinases/genética , Especificidade da Espécie , Virulência/genética
16.
Nat Commun ; 10(1): 2213, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101826

RESUMO

Spiradenoma and cylindroma are distinctive skin adnexal tumors with sweat gland differentiation and potential for malignant transformation and aggressive behaviour. We present the genomic analysis of 75 samples from 57 representative patients including 15 cylindromas, 17 spiradenomas, 2 cylindroma-spiradenoma hybrid tumors, and 24 low- and high-grade spiradenocarcinoma cases, together with morphologically benign precursor regions of these cancers. We reveal somatic or germline alterations of the CYLD gene in 15/15 cylindromas and 5/17 spiradenomas, yet only 2/24 spiradenocarcinomas. Notably, we find a recurrent missense mutation in the kinase domain of the ALPK1 gene in spiradenomas and spiradenocarcinomas, which is mutually exclusive from mutation of CYLD and can activate the NF-κB pathway in reporter assays. In addition, we show that high-grade spiradenocarcinomas carry loss-of-function TP53 mutations, while cylindromas may have disruptive mutations in DNMT3A. Thus, we reveal the genomic landscape of adnexal tumors and therapeutic targets.


Assuntos
Carcinoma Adenoide Cístico/genética , Enzima Desubiquitinante CYLD/genética , Proteínas Quinases/genética , Neoplasias das Glândulas Sudoríparas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Adenoide Cístico/patologia , Estudos de Coortes , DNA (Citosina-5-)-Metiltransferases/genética , Análise Mutacional de DNA , Feminino , Humanos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Domínios Proteicos/genética , Neoplasias das Glândulas Sudoríparas/patologia , Glândulas Sudoríparas/patologia , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do Exoma
17.
Microb Pathog ; 133: 103560, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145981

RESUMO

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8- and CD3+CD8+CD4- T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ±â€¯2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis.


Assuntos
Imunização , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/prevenção & controle , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Clonagem Molecular , Citocinas/metabolismo , Feminino , Genes de Protozoários/genética , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Linfócitos/imunologia , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Baço/imunologia , Análise de Sobrevida , Toxoplasma/genética , Toxoplasmose Animal/imunologia , Vacinas de DNA/imunologia
18.
Nat Cell Biol ; 21(6): 731-742, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086261

RESUMO

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1ß release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.


Assuntos
Artrite/genética , Inflamação/genética , Fatores de Transcrição Kruppel-Like/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Animais , Artrite/induzido quimicamente , Artrite/patologia , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Mutação , NF-kappa B/genética , Necrose/genética , Necrose/patologia , Ligação Proteica , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Ubiquitina/genética
19.
BMC Genomics ; 20(1): 307, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31014245

RESUMO

BACKGROUND: Protein kinases are enzymes controlling different cellular functions. Genetic alterations often result in kinase dysregulation, making kinases a very attractive class of druggable targets in several human diseases. Existing approved drugs still target a very limited portion of the human 'kinome', demanding a broader functional knowledge of individual and co-expressed kinase patterns in physiologic and pathologic settings. The development of novel rapid and cost-effective methods for kinome screening is therefore highly desirable, potentially leading to the identification of novel kinase drug targets. RESULTS: In this work, we describe the development of KING-REX (KINase Gene RNA EXpression), a comprehensive kinome RNA targeted custom assay-based panel designed for Next Generation Sequencing analysis, coupled with a dedicated data analysis pipeline. We have conceived KING-REX for the gene expression analysis of 512 human kinases; for 319 kinases, paired assays and custom analysis pipeline features allow the evaluation of 3'- and 5'-end transcript imbalances as readout for the prediction of gene rearrangements. Validation tests on cell line models harboring known gene fusions demonstrated a comparable accuracy of KING-REX gene expression assessment as in whole transcriptome analyses, together with a robust detection of transcript portion imbalances in rearranged kinases, even in complex RNA mixtures or in degraded RNA. CONCLUSIONS: These results support the use of KING-REX as a rapid and cost effective kinome investigation tool in the field of kinase target identification for applications in cancer biology and other human diseases.


Assuntos
Perfilação da Expressão Gênica/métodos , Proteínas Quinases/genética , Fusão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Quinases/metabolismo , Estabilidade de RNA
20.
Int J Mol Sci ; 20(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939810

RESUMO

Lysin-motif receptor-like kinase PsK1 is involved in symbiosis initiation and the maintenance of infection thread (IT) growth and bacterial release in pea. We verified PsK1 specificity in relation to the Nod factor structure using k1 and rhizobial mutants. Inoculation with nodO and nodE nodO mutants significantly reduced root hair deformations, curling, and the number of ITs in k1-1 and k1-2 mutants. These results indicated that PsK1 function may depend on Nod factor structures. PsK1 with replacement in kinase domain and PsSYM10 co-production in Nicotiana benthamiana leaves did not induce a hypersensitive response (HR) because of the impossibility of signal transduction into the cell. Replacement of P169S in LysM3 domain of PsK1 disturbed the extracellular domain (ECD) interaction with PsSYM10's ECD in Y2H system and reduced HR during the co-production of full-length PsK1 and PsSYM0 in N. benthamiana. Lastly, we explored the role of PsK1 in symbiosis with arbuscular mycorrhizal (AM) fungi; no significant differences between wild-type plants and k1 mutants were found, suggesting a specific role of PsK1 in legume⁻rhizobial symbiosis. However, increased sensitivity to a highly aggressive Fusarium culmorum strain was found in k1 mutants compared with the wild type, which requires the further study of the role of PsK1 in immune response regulation.


Assuntos
Variação Estrutural do Genoma , Ervilhas/genética , Proteínas de Plantas/genética , Proteínas Quinases/genética , Simbiose , Fusarium/patogenicidade , Micorrizas/genética , Ervilhas/microbiologia , Proteínas de Plantas/química , Domínios Proteicos , Proteínas Quinases/química , Rhizobium/patogenicidade , Tabaco/genética , Tabaco/microbiologia
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