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1.
Am Heart J ; 225: 108-119, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32480058

RESUMO

INTRODUCTION: Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined. METHODS AND RESULTS: We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults. Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%-3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-16.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6×10-5; U.S. cohort, P = 2.2×10-13). CONCLUSION: Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy.


Assuntos
Cardiomiopatias/genética , Heterozigoto , Mutação com Perda de Função , Proteínas Musculares/genética , Mutação de Sentido Incorreto , Proteínas Quinases/genética , Anormalidades Múltiplas/genética , Adulto , Idade de Início , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/fisiopatologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Hipertrófica/genética , Criança , Pré-Escolar , Cromossomos Humanos Par 15/genética , Ecocardiografia , Eletrocardiografia , Humanos , Lactente , Fenótipo
2.
Am J Physiol Heart Circ Physiol ; 318(6): H1509-H1515, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32383995

RESUMO

Protein kinases play an integral role in cardiac development, function, and disease. Recent experimental and clinical data have implied that protein kinases belonging to a family of atypical α-protein kinases, including α-protein kinase 2 (ALPK2), are important for regulating cardiac development and maintaining function via regulation of WNT signaling. A recent study in zebrafish reported that loss of ALPK2 leads to severe cardiac defects; however, the relevance of ALPK2 has not been studied in a mammalian animal model. To assess the role of ALPK2 in the mammalian heart, we generated two independent global Alpk2-knockout (Alpk2-gKO) mouse lines, using CRISPR/Cas9 technology. We performed physiological and biochemical analyses of Alpk2-gKO mice to determine the functional, morphological, and molecular consequences of Alpk2 deletion at the organismal level. We found that Alpk2-gKO mice exhibited normal cardiac function and morphology up to one year of age. Moreover, we did not observe altered WNT signaling in neonatal Alpk2-gKO mouse hearts. In conclusion, Alpk2 is dispensable for cardiac development and function in the murine model. Our results suggest that Alpk2 is a rapidly evolving gene that lost its essential cardiac functions in mammals.NEW & NOTEWORTHY Several studies indicated the importance of ALPK2 for cardiac function and development. A recent study in zebrafish report that loss of ALPK2 leads to severe cardiac defects. In contrast, murine Alpk2-gKO models developed in this work display no overt cardiac phenotype. Our results suggest ALPK2, as a rapidly evolving gene, lost its essential cardiac functions in mammals.


Assuntos
Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Proteínas Quinases/genética , Animais , Coração/fisiologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Proteínas Quinases/metabolismo
3.
Nature ; 581(7807): 199-203, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32404997

RESUMO

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Imunidade Vegetal/imunologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Arabidopsis/enzimologia , Endocitose , Ligantes , Padrões Moleculares Associados a Patógenos/imunologia , Fosforilação , Proteínas Quinases/metabolismo
4.
Plant Mol Biol ; 103(4-5): 581-596, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32409993

RESUMO

KEY MESSAGE: N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects. Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N154-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Polissacarídeos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Alcaloides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Modelos Moleculares , Oligossacarídeos/metabolismo , Plantas Geneticamente Modificadas , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Plântula , Sementes/citologia , Sementes/metabolismo , Transdução de Sinais/genética
5.
Cell Prolif ; 53(6): e12817, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32396704

RESUMO

OBJECTIVES: Cadmium (Cd) induces mitophagy in neuronal cells, but the underlying mechanisms remain unknown. In this study, we aimed to investigate these mechanisms. MATERIALS AND METHODS: The effects of Cd on the mitophagy in rat pheochromocytoma PC12 cells were detected, and the role of PINK1/Parkin pathway in Cd-induced mitophagy was also analysed by using PINK1 siRNA. In order to explore the relationship between AMPK and PINK1/Parkin in Cd-induced mitophagy in PC12 cells, the CRISPR-Cas9 system was used to knock down AMPK expression. RESULTS: The results showed that Cd treatment triggered a significant increase in mitophagosome formation and the colocalization of mitochondria and lysosomes, which was further proved by the colocalization of LC3 puncta and its receptors NDP52 or P62 with mitochondria in PC12 cells. Moreover, an accumulation of PINK1 and Parkin was found in mitochondria. Additionally, upon PINK1 knock-down using PINK1 siRNA, Cd-induced mitophagy was efficiently suppressed. Interestingly, chemical or genetic reversal of AMPK activation: (a) significantly inhibited the activation of mitophagy and (b) promoted NLRP3 activation by inhibiting PINK/Parkin translocation. CONCLUSIONS: These results suggest that Cd induces mitophagy via the PINK/Parkin pathway following AMPK activation in PC12 cells. Targeting the balanced activity of AMPK/PINK1/Parkin-mediated mitophagy signalling may be a potential therapeutic approach to treat Cd-induced neurotoxicity.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cádmio/farmacologia , Mitofagia/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Ativação Enzimática , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células PC12 , Ratos
6.
PLoS Genet ; 16(5): e1008722, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392214

RESUMO

To survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins, depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA's cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Salmonella enterica/genética , Salmonella enterica/metabolismo , Transativadores/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo
7.
Parasitol Res ; 119(5): 1675-1681, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32236711

RESUMO

Toxoplasma gondii can infect virtually all warm-blooded animals, including humans. It can differentiate between rapidly replicating tachyzoites that cause acute infection and slowly growing bradyzoites in tissue cysts. Treatment options for toxoplasmosis are challenging because current therapies cannot eradicate the latent T. gondii infection that is mainly caused by the bradyzoite forms. Accordingly, recurrence of infection is a problem for immunocompromised patients and congenitally infected patients. Protein kinases have been widely studied in eukaryotic cells, and while little is known about signaling in Toxoplasma infection, it is likely that protein kinases play a key role in parasite proliferation, differentiation, and probably invasion. To identify optimized new kinase inhibitors for drug development against T. gondii, we screened a library of kinase inhibitor compounds for anti-Toxoplasma activity and host cell cytotoxicity. Pyrimethamine served as a positive control and 0.5% DMSO was used as a negative control. Among the 80 compounds screened, 6 compounds demonstrated ≥ 80% parasite growth inhibition at concentrations at which 5 compounds did not suppress host cell viability, while 3 kinase inhibitors (Bay 11-7082, Tyrphostin AG 1295 and PD-98059) had suppressive effects individually on parasite growth and host cell invasion, but did not strongly induce bradyzoite formation.


Assuntos
Antiprotozoários/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/tratamento farmacológico , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/farmacologia , Humanos , Nitrilos/farmacologia , Pirimetamina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/patogenicidade , Toxoplasmose/patologia , Tirfostinas/farmacologia
8.
Plant Physiol Biochem ; 151: 650-658, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32339912

RESUMO

In plants, basic leucine zipper (bZIP) transcription factors (TFs) participate in various biological processes such as development and stress responses. But the molecular mechanism of wheat bZIP TFs modulating abscisic acid (ABA) biosynthesis is unknown. In this study, we demonstrated the expressions of three bZIP TF genes TabZIP8, 9, 13, were regulated by Triticum aestivum calcium (Ca2+)-dependent protein kinase 9-1 (TaCDPK9-1) and they took part in ABA biosynthesis in wheat roots under salt stress. We first isolated TabZIP8, 9, 13 and TaCDPK9-1 from wheat. TabZIP8, 9, 13 genes transcripts were strongly induced by salt stress, but salt-induced TabZIP8, 9, 13 transcriptions were drastically impaired by Ca2+ channel blocker LaCl3. TaCDPK9-1 kinase could interact with TabZIP8, 9, 13 TFs through yeast two-hybrid assay. Next, the expression levels of salt-induced wheat 9-cis-epoxycarotenoid dioxygenase1, 2 (TaNCED1,2) encoding a key enzyme controlling ABA production and salt-induced ABA content were found to be decreased under LaCl3 treatment, and yeast one-hybrid experiment revealed TabZIP8, 9, 13 could bind to the ABA-responsive elements (ABREs) and the promoter sequence of TaNCED2 gene. Together, our results suggest that salt stress-induced ABA accumulation is mediated by TabZIP8, 9, 13, which are adjusted by TaCDPK9-1 in wheat roots.


Assuntos
Ácido Abscísico , Proteínas de Plantas , Proteínas Quinases , Estresse Fisiológico , Triticum , Ácido Abscísico/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Proteínas Quinases/metabolismo , Cloreto de Sódio/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Triticum/genética , Triticum/metabolismo
9.
Am J Physiol Heart Circ Physiol ; 318(5): H1256-H1271, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32223553

RESUMO

Despite decades of research on the pathophysiology of myocardial stunning, protein changes and/or phosphorylation status underlying alterations in cardiac function/structure remain inadequately understood. Here, we utilized comprehensive and quantitative proteomic and phosphoproteomic approaches to explore molecular mechanisms of myocardial stunning in swine. The closed-chest swine (n = 5 pigs) were subjected to a 10-min left anterior descending coronary artery (LAD) occlusion producing regional myocardial stunning. Tissues from the ischemic LAD region and a remote nonischemic area of the left ventricle were collected 1 h after reperfusion. Ion current-based proteomics (IonStar) and quantitative phosphoproteomics were employed in parallel to identify alterations in protein level and site-specific phosphorylation changes. A novel swine heart protein database exhibiting high accuracy and low redundancy was developed here to facilitate comprehensive study. Further informatic investigations identified potential protein-protein interactions in stunned myocardium. In total, we quantified 2,099 protein groups and 4,699 phosphorylation sites with only 0.4% missing values. Proteomic analyses revealed downregulation of contractile function and extracellular matrix remodeling. Meanwhile, alterations in phosphorylation linked with contractile dysfunction and apoptotic cell death were uncovered. NetworKIN/STRING analysis predicted regulatory kinases responsible for altered phosphosites, such as protein kinase C-mediated phosphorylation of cardiac troponin I-S199 and CaMKII-mediated phosphorylation of phospholamban-T17. In summary, the ion current-based proteomics and phosphoproteomics reliably identified novel alterations in protein content and phosphorylation contributing to contractile dysfunction, extracellular matrix (ECM) damage, and programmed cell death in stunned myocardium, which corroborate well with our physiological observations. Moreover, this work developed a comprehensive database of the swine heart proteome, a highly valuable resource for future translational research in porcine models with cardiovascular diseases.NEW & NOTEWORTHY We first used ion current-based proteomics and phosphoproteomics to reliably identify novel alterations in protein expression and phosphorylation contributing to contractile dysfunction, extracellular matrix (ECM) damage, and programmed cell death in stunned myocardium and developed a comprehensive swine heart-specific proteome database, which provides a valuable resource for future research in porcine models of cardiovascular diseases.


Assuntos
Doença das Coronárias/metabolismo , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Potenciais de Ação , Animais , Doença das Coronárias/genética , Doença das Coronárias/fisiopatologia , Masculino , Contração Miocárdica , Fosfoproteínas/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteoma/genética , Suínos
10.
PLoS One ; 15(3): e0230819, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32231398

RESUMO

STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.


Assuntos
Antineoplásicos/farmacologia , Biologia Computacional , Fator de Transcrição STAT3/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Fatores de Tempo
11.
Curr Opin Plant Biol ; 55: 52-59, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32259743

RESUMO

SUCROSE-NON-FERMENTING1-RELATED KINASE1 (SnRK1) belongs to a family of protein kinases that originated in the earliest eukaryotes and plays a central role in energy and metabolic homeostasis. Trehalose 6-phosphate (Tre6P) is the intermediate of trehalose biosynthesis, and has even more ancient roots, being found in all three domains of life - Archaea, Bacteria and Eukarya. In plants, the function of SnRK1 has diverged from its orthologues in fungi and animals, evolving new roles in signalling of nutrient status and abiotic stress. Tre6P has also acquired a novel function in plants as a signal and homeostatic regulator of sucrose, the dominant sugar in plant metabolism. These two ancient pathways have converged in a unique way in plants, enabling them to coordinate their metabolism, growth, and development with their environment, which is essential for their autotrophic and sessile lifestyle.


Assuntos
Fosfatos Açúcares , Trealose , Animais , Regulação da Expressão Gênica de Plantas , Fosfatos , Plantas , Proteínas Quinases , Sacarose
12.
Proc Natl Acad Sci U S A ; 117(15): 8468-8475, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234780

RESUMO

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.


Assuntos
Materiais Biomiméticos/farmacologia , Membrana Celular/metabolismo , Domínio de Fibronectina Tipo III , Necrose , Oligopeptídeos/farmacologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Cristalografia por Raios X , Humanos , Fosforilação , Conformação Proteica , Proteínas Quinases/química , Multimerização Proteica , Transporte Proteico
13.
Nat Commun ; 11(1): 1496, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198415

RESUMO

The ability to grow at moderate acidic conditions (pH 4.0-5.0) is important to Escherichia coli colonization of the host's intestine. Several regulatory systems are known to control acid resistance in E. coli, enabling the bacteria to survive under acidic conditions without growth. Here, we characterize an acid-tolerance response (ATR) system and its regulatory circuit, required for E. coli exponential growth at pH 4.2. A two-component system CpxRA directly senses acidification through protonation of CpxA periplasmic histidine residues, and upregulates the fabA and fabB genes, leading to increased production of unsaturated fatty acids. Changes in lipid composition decrease membrane fluidity, F0F1-ATPase activity, and improve intracellular pH homeostasis. The ATR system is important for E. coli survival in the mouse intestine and for production of higher level of 3-hydroxypropionate during fermentation. Furthermore, this ATR system appears to be conserved in other Gram-negative bacteria.


Assuntos
Tolerância a Medicamentos/fisiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Fermentação , Regulação Bacteriana da Expressão Gênica , Homeostase , Hidroliases/genética , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Intestinos/microbiologia , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Fluidez de Membrana , Lipídeos de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases/metabolismo , Transcrição Genética
14.
Proc Natl Acad Sci U S A ; 117(11): 6223-6230, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123097

RESUMO

All multicellular organisms keep a balance between sink and source activities by controlling nutrient transport at strategic positions. In most plants, photosynthetically produced sucrose is the predominant carbon and energy source, whose transport from leaves to carbon sink organs depends on sucrose transporters. In the model plant Arabidopsis thaliana, transport of sucrose into the phloem vascular tissue by SUCROSE TRANSPORTER 2 (SUC2) sets the rate of carbon export from source leaves, just like the SUC2 homologs of most crop plants. Despite their importance, little is known about the proteins that regulate these sucrose transporters. Here, identification and characterization of SUC2-interaction partners revealed that SUC2 activity is regulated via its protein turnover rate and phosphorylation state. UBIQUITIN-CONJUGATING ENZYME 34 (UBC34) was found to trigger turnover of SUC2 in a light-dependent manner. The E2 enzyme UBC34 could ubiquitinate SUC2 in vitro, a function generally associated with E3 ubiquitin ligases. ubc34 mutants showed increased phloem loading, as well as increased biomass and yield. In contrast, mutants of another SUC2-interaction partner, WALL-ASSOCIATED KINASE LIKE 8 (WAKL8), showed decreased phloem loading and growth. An in vivo assay based on a fluorescent sucrose analog confirmed that SUC2 phosphorylation by WAKL8 can increase transport activity. Both proteins are required for the up-regulation of phloem loading in response to increased light intensity. The molecular mechanism of SUC2 regulation elucidated here provides promising targets for the biotechnological enhancement of source strength.


Assuntos
Arabidopsis/fisiologia , Sequestro de Carbono , Carbono/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutação , Floema/metabolismo , Fosforilação/fisiologia , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia
15.
Nat Commun ; 11(1): 1361, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170184

RESUMO

Grana are a characteristic feature of higher plants' thylakoid membranes, consisting of stacks of appressed membranes enriched in Photosystem II (PSII) and associated light-harvesting complex II (LHCII) proteins, together forming the PSII-LHCII supercomplex. Grana stacks undergo light-dependent structural changes, mainly by reorganizing the supramolecular structure of PSII-LHCII supercomplexes. LHCII is vital for grana formation, in which also PSII-LHCII supercomplexes are involved. By combining top-down and crosslinking mass spectrometry we uncover the spatial organization of paired PSII-LHCII supercomplexes within thylakoid membranes. The resulting model highlights a basic molecular mechanism whereby plants maintain grana stacking at changing light conditions. This mechanism relies on interactions between stroma-exposed N-terminal loops of LHCII trimers and Lhcb4 subunits facing each other in adjacent membranes. The combination of light-dependent LHCII N-terminal trimming and extensive N-terminal α-acetylation likely affects interactions between pairs of PSII-LHCII supercomplexes across the stromal gap, ultimately mediating membrane folding in grana stacks.


Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Plantas/metabolismo , Proteínas Quinases/metabolismo , Tilacoides/metabolismo , Proteínas de Ligação à Clorofila/metabolismo , Embriófitas , Luz , Complexos de Proteínas Captadores de Luz/química , Espectrometria de Massas/métodos , Modelos Moleculares , Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Proteínas Quinases/química , Proteômica
16.
Arterioscler Thromb Vasc Biol ; 40(5): 1155-1167, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32212851

RESUMO

OBJECTIVES: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. CONCLUSIONS: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.


Assuntos
Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Colesterol/metabolismo , Células Espumosas/enzimologia , Macrófagos Peritoneais/enzimologia , Placa Aterosclerótica , Proteínas Quinases/deficiência , Animais , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Modelos Animais de Doenças , Endossomos/metabolismo , Feminino , Células Espumosas/patologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos Knockout para ApoE , Necroptose , Necrose , Oligonucleotídeos Antissenso/administração & dosagem , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais
17.
PLoS One ; 15(3): e0221006, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32187190

RESUMO

Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.


Assuntos
Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Músculos/anatomia & histologia , Músculos/metabolismo , Sistema Nervoso/anatomia & histologia , Sistema Nervoso/metabolismo , Proteínas Quinases/isolamento & purificação , Animais , Padronização Corporal , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Olho/embriologia , Larva/metabolismo , Masculino , Músculos/citologia , Sistema Nervoso/citologia , Junção Neuromuscular/metabolismo , Tamanho do Órgão , Fosforilação , Sinapses/metabolismo
18.
PLoS Negl Trop Dis ; 14(3): e0008115, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203512

RESUMO

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a 'footprint' of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.


Assuntos
Proteínas de Helminto/metabolismo , Fosfoproteínas/metabolismo , Proteoma/análise , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Feminino , Proteínas de Helminto/genética , Masculino , Peptídeos/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteínas Quinases , Processamento de Proteína Pós-Traducional , Schistosoma mansoni/genética , Transdução de Sinais
19.
PLoS Genet ; 16(3): e1008561, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32134924

RESUMO

Intraflagellar transport (IFT) is required for ciliary assembly and maintenance. While disruption of IFT may trigger ciliary disassembly, we show here that IFT mediated transport of a CDK-like kinase ensures proper ciliary disassembly. Mutations in flagellar shortening 2 (FLS2), encoding a CDK-like kinase, lead to retardation of cilia resorption and delay of cell cycle progression. Stimulation for ciliary disassembly induces gradual dephosphorylation of FLS2 accompanied with gradual inactivation. Loss of FLS2 or its kinase activity induces early onset of kinesin13 phosphorylation in cilia. FLS2 is predominantly localized in the cell body, however, it is transported to cilia upon induction of ciliary disassembly. FLS2 directly interacts with IFT70 and loss of this interaction inhibits its ciliary transport, leading to dysregulation of kinesin13 phosphorylation and retardation of ciliary disassembly. Thus, this work demonstrates that IFT plays active roles in controlling proper ciliary disassembly by transporting a protein kinase to cilia to regulate a microtubule depolymerizer.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteína Quinase CDC2/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Transporte Biológico/fisiologia , Ciclo Celular/fisiologia , Flagelos/metabolismo , Fosforilação/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/fisiologia
20.
PLoS One ; 15(3): e0228385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134954

RESUMO

Our earlier studies proved that RIPK3-mediated necroptosis might be an important mode of renal tubular cell death in rats with chronic renal injury and the necroptotic cell death can be triggered by tumor necrosis factor-α (TNF-α) in vitro, but the triggering role of angiotensin II (AngII), which exerts notable effects on renal cells for the initiation and progression of renal tubulointerstitial fibrosis, is largely unknown. Here, we identified the presence of necroptotic cell death in the tubular cells of AngII-induced chronic renal injury and fibrosis mice and assessed the percentage of necroptotic renal tubular cell death with the disruption of this necroptosis by the addition of necrostatin-1 (Nec-1). Furthermore, the observation was further confirmed in HK-2 cells treated with AngII and RIPK1/3 or MLKL inhibitors. The detection of Fas and FasL proteins led us to investigate the contribution of the Fas/FasL signaling pathway to AngII-induced necroptosis. Disruption of FasL decreased the percentage of necroptotic cells, suggesting that Fas and FasL are likely key signal molecules in the necroptosis of HK-2 cells induced by AngII. Our data suggest that AngII exposure might trigger RIPK3-MLKL-mediated necroptosis in renal tubular epithelial cells by activating the Fas/FasL signaling pathway in vivo and in vitro.


Assuntos
Angiotensina II/farmacologia , Proteína Ligante Fas/metabolismo , Túbulos Renais/citologia , Necroptose/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptor fas/metabolismo , Animais , Linhagem Celular , Fibrose , Humanos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos
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