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1.
Science ; 368(6489)2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32327570

RESUMO

Protein quality control is essential for the proper function of cells and the organisms that they make up. The resulting loss of proteostasis, the processes by which the health of the cell's proteins is monitored and maintained at homeostasis, is associated with a wide range of age-related human diseases. Here, we highlight how the integrated stress response (ISR), a central signaling network that responds to proteostasis defects by tuning protein synthesis rates, impedes the formation of long-term memory. In addition, we address how dysregulated ISR signaling contributes to the pathogenesis of complex diseases, including cognitive disorders, neurodegeneration, cancer, diabetes, and metabolic disorders. The development of tools through which the ISR can be modulated promises to uncover new avenues to diminish pathologies resulting from it for clinical benefit.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteostase , Estresse Fisiológico , Fatores de Complexo Ternário/metabolismo , Acetamidas/química , Acetamidas/farmacologia , Animais , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Humanos , Imunidade , Doenças Metabólicas/metabolismo , Camundongos , Neoplasias/metabolismo , Fosfotransferases/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(3): 1485-1495, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31911473

RESUMO

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.


Assuntos
Proteínas de Escherichia coli/química , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Metiltransferases de Proteína/química , Metiltransferases de Proteína/genética , Metiltransferases de Proteína/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
3.
BMC Plant Biol ; 20(1): 26, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31948398

RESUMO

BACKGROUND: The receptor-like kinase FEROINA (FER) plays a crucial role in controlling plant vegetative growth partially by sensing the rapid alkalinization factor (RALF) peptide. However, the role of RALF1-FER in the vegetative-reproductive growth transition remains unknown. Here, we analyze the mechanism through which FER affects the flowering time in Arabidopsis. RESULTS: We found that the FER mRNA levels exhibit an oscillating pattern with a diurnal rhythm and that the clock oscillator CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) up-regulates the expression of FER by associating with its chromatin. In addition, FER expression is regulated by clock genes, and FER also modulates the expression patterns of clock genes. Consistent with its gene expression pattern, FER positively regulates flowering by modulating the transcript accumulation and mRNA alternative splicing of certain flowering-related genes, including FLOWERING LOCUS C (FLC) and its homolog MADS AFFECTING FLOWERING (MAF). However, the RALF1 ligand negatively regulates flowering compared with FER. CONCLUSIONS: We found that FER, which is up-regulated by CCA1, controls the flowering time by regulating the transcript accumulation and mRNA alternative splicing (AS) of some important flowering genes, and these findings link FER to the floral transition.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flores/genética , Fosfotransferases/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fosfotransferases/metabolismo , Fatores de Transcrição/metabolismo
4.
Invest Ophthalmol Vis Sci ; 61(1): 1, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31995153

RESUMO

Purpose: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. Methods: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. Results: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. Conclusions: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Fosfotransferases/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas tau/metabolismo , Animais , Western Blotting , Células Cultivadas , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Regulação para Baixo , Técnica Indireta de Fluorescência para Anticorpo , Ácido Glutâmico/toxicidade , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Roscovitina/farmacologia , Transfecção , Enzimas Ativadoras de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
5.
Chem Commun (Camb) ; 55(89): 13330-13341, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31633708

RESUMO

DNA-encoded library (DEL) screening has emerged as an important method for early stage drug and probe molecule discovery. The vast majority of screens using DELs have been relatively simple binding assays. The library is incubated with a target molecule, which is almost always a protein, and the DNAs that remain associated with the target after thorough washing are amplified and deep sequenced to reveal the chemical structures of the ligands they encode. Recently however, a number of different screening formats have been introduced that demand more than simple binding. These include a format that demands hits exhibit high selectivity for target vs. off-targets, a protocol to screen for enzyme inhibitors and another to identify organocatalysts in a DEL. These and other novel assay formats are reviewed in this article. We also consider some of the most significant remaining challenges in DEL assay development.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Biblioteca Gênica , Ligantes , Biblioteca de Peptídeos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Ligação Proteica/efeitos dos fármacos
6.
Nat Neurosci ; 22(11): 1806-1819, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636448

RESUMO

Prediabetes and Alzheimer's disease both increase in prevalence with age. The former is a risk factor for the latter, but a mechanistic linkage between them remains elusive. We show that prediabetic serum hyperinsulinemia is reflected in the cerebrospinal fluid and that this chronically elevated insulin renders neurons resistant to insulin. This leads to abnormal electrophysiological activity and other defects. In addition, neuronal insulin resistance reduces hexokinase 2, thus impairing glycolysis. This hampers the ubiquitination and degradation of p35, favoring its cleavage to p25, which hyperactivates CDK5 and interferes with the GSK3ß-induced degradation of ß-catenin. CDK5 contributes to neuronal cell death while ß-catenin enters the neuronal nucleus and re-activates the cell cycle machinery. Unable to successfully divide, the neuron instead enters a senescent-like state. These findings offer a direct connection between peripheral hyperinsulinemia, as found in prediabetes, age-related neurodegeneration and cognitive decline. The implications for neurodegenerative conditions such as Alzheimer's disease are described.


Assuntos
Envelhecimento/fisiologia , Ciclo Celular/fisiologia , Senescência Celular/fisiologia , Hiperinsulinismo/fisiopatologia , Resistência à Insulina/fisiologia , Neurônios/fisiologia , Animais , Morte Celular/fisiologia , Senescência Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Hexoquinase/metabolismo , Hiperinsulinismo/líquido cefalorraquidiano , Potenciais Pós-Sinápticos Inibidores/fisiologia , Insulina/farmacologia , Liraglutida/farmacologia , Masculino , Aprendizagem em Labirinto/fisiologia , Metformina/farmacologia , Camundongos , Neurônios/metabolismo , Fosfotransferases/metabolismo , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitinação/fisiologia , beta Catenina/metabolismo
7.
Endocrinology ; 160(11): 2692-2708, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31433456

RESUMO

Human prostate stem and progenitor cells express estrogen receptor (ER)α and ERß and exhibit proliferative responses to estrogens. In this study, membrane-initiated estrogen signaling was interrogated in human prostate stem/progenitor cells enriched from primary epithelial cultures and stem-like cell lines from benign and cancerous prostates. Subcellular fractionation and proximity ligation assays localized ERα and ERß to the cell membrane with caveolin-1 interactions. Exposure to 17ß-estradiol (E2) for 15 to 60 minutes led to sequential phosphorylation of signaling molecules in MAPK and AKT pathways, IGF1 receptor, epidermal growth factor receptor, and ERα, thus documenting an intact membrane signalosome that activates diverse downstream cascades. Treatment with an E2-dendrimer conjugate or ICI 182,870 validated E2-mediated actions through membrane ERs. Overexpression and knockdown of ERα or ERß in stem/progenitor cells identified pathway selectivity; ERα preferentially activated AKT, whereas ERß selectively activated MAPK cascades. Furthermore, prostate cancer stem-like cells expressed only ERß, and brief E2 exposure activated MAPK but not AKT cascades. A gene subset selectively regulated by nongenomic E2 signaling was identified in normal prostate progenitor cells that includes BGN, FOSB, FOXQ1, and MAF. Membrane-initiated E2 signaling rapidly modified histone methyltransferases, with MLL1 cleavage observed downstream of phosphorylated AKT and EZH2 phosphorylation downstream of MAPK signaling, which may jointly modify histones to permit rapid gene transcription. Taken together, the present findings document ERα and ERß membrane-initiated signaling in normal and cancerous human prostate stem/progenitor cells with differential engagement of downstream effectors. These signaling pathways influence normal prostate stem/progenitor cell homeostasis and provide novel therapeutic sites to target the elusive prostate cancer stem cell population.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Células-Tronco Neoplásicas/metabolismo , Próstata/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Histona Metiltransferases/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Fosforilação , Fosfotransferases/metabolismo , Próstata/citologia
8.
BMC Genomics ; 20(1): 674, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455217

RESUMO

BACKGROUND: Ethylene signal transduction in plants is conducted by the two-component system (TCS) which consists of histidine kinase (HK), histidine phosphotransferase (HPT) and response regulators (RRs). This system plays an important role in signal transduction during various cellular processes, including fruit ripening and response to multiple environmental cues. Though members of TCS have been identified in a few plants, no detailed analysis has been carried out in banana. RESULTS: Through genome-wide analysis, we identified a total of 80 (25 HK, 10 HPT and 45 RR) and 72 (25 HK, 5 HPT and 42 RR) TCS genes in Musa acuminata and Musa balbisiana respectively. The analysis of identified genes revealed that most of the genes are highly conserved however; there are subtle divergences among various members. Comparative expression analysis revealed an involvement of a set of TCS members during banana fruit ripening. Co-expression network analysis identified a working TCS module with direct interactions of HK-HPT and RR members. The molecular dynamics analysis of TCS module showed a significant change in structural trajectories of TCS proteins in the presence of ethylene. Analysis suggests possible interactions between the HK-HPTs and RRs as well as other members leading to banana fruit ripening. CONCLUSIONS: In this study, we identified and compared the members of TCS gene family in two banana species and showed their diversity, within groups on the basis of whole-genome duplication events. Our analysis showed that during banana fruit ripening TCS module plays a crucial role. We also demonstrated a possible interaction mechanism of TCS proteins in the presence and absence of ethylene by molecular dynamics simulations. These findings will help in understanding the functional mechanism of TCS proteins in plants in different conditions.


Assuntos
Etilenos/metabolismo , Musa/genética , Musa/metabolismo , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Histidina Quinase/metabolismo , Fosfotransferases/metabolismo , Filogenia , Domínios e Motivos de Interação entre Proteínas , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais
9.
Nat Commun ; 10(1): 3354, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350417

RESUMO

How microbes dynamically coordinate uptake and simultaneous utilization of nutrients in complex nutritional ecosystems is still an open question. Here, we develop a constraint-based modeling approach that exploits non-targeted exo-metabolomics data to unravel adaptive decision-making processes in dynamic nutritional environments. We thereby investigate metabolic adaptation of Escherichia coli to continuously changing conditions during batch growth in complex medium. Unexpectedly, model-based analysis of time resolved exo-metabolome data revealed that fastest growth coincides with preferred catabolism of amino acids, which, in turn, reduces glucose uptake and increases acetate overflow. We show that high intracellular levels of the amino acid degradation metabolites pyruvate and oxaloacetate can directly inhibit the phosphotransferase system (PTS), and reveal their functional role in mediating regulatory decisions for uptake and catabolism of alternative carbon sources. Overall, the proposed methodology expands the spectrum of possible applications of flux balance analysis to decipher metabolic adaptation mechanisms in naturally occurring habitats and diverse organisms.


Assuntos
Aminoácidos/metabolismo , Escherichia coli/metabolismo , Glucose/metabolismo , Acetatos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácido Oxaloacético/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Ácido Pirúvico/metabolismo
10.
Nature ; 572(7768): 270-274, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31291642

RESUMO

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment1. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides2 have previously been proposed as ligands for several members of the CrRLK1L family1. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfotransferases/metabolismo , Proteínas de Arabidopsis/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Modelos Moleculares , Mutagênese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfotransferases/genética , Maleabilidade , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica
11.
PLoS Pathog ; 15(7): e1007900, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31269090

RESUMO

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.


Assuntos
Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Fosfotransferases/imunologia , Fosfotransferases/metabolismo , Imunidade Vegetal , Acetilação , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Modelos Imunológicos , Mutação , Fosfotransferases/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidade
12.
Microbiol Res ; 223-225: 63-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178053

RESUMO

The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) catalyzes the translocation of sugar substrates with their concomitant phosphorylation in bacteria. In addition to its intrinsic role in sugar transport and metabolism, numerous recent studies report the versatility of the PTS to interconnect energy and signal transduction in response to sugar availability. In this study, the role of PTS in Salmonella virulence regulation was explored. To decipher the regulatory network coordinated by the PTS during Salmonella infection, a transcriptomic approach was applied to a transposon insertion mutant with defective expression of ptsI and crr, which encode enzyme I and enzyme IIAGlc of the PTS, respectively. There were 114 differentially expressed genes (DEGs) exhibiting two-fold or higher expression changes in the transposon mutant strain, with 13 up-regulated genes versus 101 down-regulated genes. One-third of the DEGs were associated with energy production and carbohydrate/amino acid metabolism pathways, implicating the prominent role of the PTS in carbohydrate transport. With regard to regulation of virulence, the tested mutant decreased the expression of genes associated with quorum sensing, Salmonella pathogenicity islands, flagella, and the PhoPQ regulon. We investigated the possibility of PTS-mediated regulation of virulence determinants identified in the transcriptomic analysis and proposed a regulatory circuit orchestrated by the PTS in Salmonella infection of host cells. These results suggest that Salmonella divergently controls virulence attributes in accordance with the availability of carbohydrates in the environment.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fosfoenolpiruvato/metabolismo , Fosfotransferases/metabolismo , Salmonella/genética , Salmonella/metabolismo , Fatores de Virulência/genética , Transporte Biológico , Elementos de DNA Transponíveis , Flagelos/genética , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Mutação , Fosforilação , Regulon , Salmonella/patogenicidade , Infecções por Salmonella , Salmonella typhimurium/genética , Transdução de Sinais , Transcriptoma , Sistemas de Secreção Tipo III/genética , Virulência/genética
13.
Nature ; 569(7757): 509-513, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31068699

RESUMO

A universal gain-of-function approach for selective and temporal control of protein activity in living systems is crucial to understanding dynamic cellular processes. Here we report development of a computationally aided and genetically encoded proximal decaging (hereafter, CAGE-prox) strategy that enables time-resolved activation of a broad range of proteins in living cells and mice. Temporal blockage of protein activity was computationally designed and realized by genetic incorporation of a photo-caged amino acid in proximity to the functional site of the protein, which can be rapidly removed upon decaging, resulting in protein re-activation. We demonstrate the wide applicability of our method on diverse protein families, which enabled orthogonal tuning of cell signalling and immune responses, temporal profiling of proteolytic substrates upon caspase activation as well as the development of protein-based pro-drug therapy. We envision that CAGE-prox will open opportunities for the gain-of-function study of proteins and dynamic biological processes with high precision and temporal resolution.


Assuntos
Sobrevivência Celular , Proteínas/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Ativação Enzimática , Mutação com Ganho de Função , Humanos , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Fosfotransferases/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/uso terapêutico , Proteínas/genética , Proteínas/imunologia , Proteínas/uso terapêutico , Proteólise , Proteômica , Transdução de Sinais , Fatores de Tempo
14.
mBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088917

RESUMO

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production.IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature's main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/genética , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Fosfotransferases/genética , Fosfotransferases/metabolismo , Antibacterianos/biossíntese , Impressões Digitais de DNA , Glucosamina/metabolismo , Glucose-6-Fosfato/metabolismo , Fosforilação , Ligação Proteica , RNA Ribossômico 16S/genética , Microbiologia do Solo
15.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036600

RESUMO

Enterococcus faecalis strains are resident intestinal bacteria associated with invasive infections, inflammatory bowel diseases, and colon cancer. Although factors promoting E. faecalis colonization of intestines are not fully known, one implicated pathway is a phosphotransferase system (PTS) in E. faecalis strain OG1RF that phosphorylates gluconate and contains the genes OG1RF_12399 to OG1RF_12402 (OG1RF_12399-12402). We hypothesize that this PTS permits growth in gluconate, facilitates E. faecalis intestinal colonization, and exacerbates colitis. We generated E. faecalis strains containing deletions/point mutations in this PTS and measured bacterial growth and PTS gene expression in minimal medium supplemented with selected carbohydrates. We show that E. faecalis upregulates OG1RF_12399 transcription specifically in the presence of gluconate and that E. faecalis strains lacking, or harboring a single point mutation in, OG1RF_12399-12402 are unable to grow in minimal medium containing gluconate. We colonized germfree wild-type and colitis-prone interleukin-10-deficient mice with defined bacterial consortia containing the E. faecalis strains and measured inflammation and bacterial abundance in the colon. We infected macrophage and intestinal epithelial cell lines with the E. faecalis strains and measured intracellular bacterial survival and proinflammatory cytokine secretion. The presence of OG1RF_12399-12402 is not required for E. faecalis colonization of the mouse intestine but is associated with an accelerated onset of experimental colitis in interleukin-10-deficient mice, altered bacterial composition in the colon, enhanced E. faecalis survival within macrophages, and increased proinflammatory cytokine secretion by colon tissue and macrophages. Further studies of bacterial carbohydrate metabolism in general, and E. faecalis PTS-gluconate in particular, during inflammation may identify new mechanisms of disease pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Colite/microbiologia , Enterococcus faecalis/enzimologia , Macrófagos/imunologia , Fosfotransferases/metabolismo , Animais , Proteínas de Bactérias/genética , Colite/genética , Colite/imunologia , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Feminino , Gluconatos/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Macrófagos/microbiologia , Masculino , Camundongos , Óperon , Fosfotransferases/genética
16.
Nat Commun ; 10(1): 1919, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015472

RESUMO

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.


Assuntos
Bacillus subtilis/genética , Cucurbitaceae/microbiologia , Matriz Extracelular/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Microbianas/genética , Pseudomonas chlororaphis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana , Fosfotransferases/genética , Fosfotransferases/metabolismo , Folhas de Planta/microbiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pseudomonas chlororaphis/crescimento & desenvolvimento , Pseudomonas chlororaphis/metabolismo , Sementes/microbiologia , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Simbiose/fisiologia , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo
17.
Microb Pathog ; 130: 131-136, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30858007

RESUMO

The objective of this study was to evaluate the productive impact of colibacillosis on laying hens and to investigate whether energetic metabolism and oxidative stress were involved in the pathogenesis of the disease. An experimental shed containing 270 laying hens of the Hy-Line lineage (32 weeks old) presented approximately 40% daily laying, and many birds presented with diarrhea and apathy followed by death. Necropsy revealed macroscopic lesions compatible with colibacillosis and infectious agent Escherichia coli was isolated from fecal samples of all birds in the infected group, as well as from tissue (ovary, liver and peritoneum). Sixteen chickens were selected for this study, divided into two groups: Control (animals without clinical alterations) and infected (with diarrhea and apathetic). E. coli isolates were subjected to the antimicrobial susceptibility testing according to the methodology approved by CLSI, 2018. This testing showed sensitivity to gentamicin, amoxicillin, norfloxacin and colistin. It was then determined that laying hens would be treated with norfloxacin (15 mg/kg) diluted in water offered at will to the birds for three days. Blood collections were performed via brachial vein after the diagnosis of E. coli (before starting treatment) and seven days after treatment. Three debilitated chickens died on the second day after initiating therapy. Before treatment, birds with clinical signs had higher levels of lipoperoxidation (LPO) and activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) than in the control group (asymptomatic animals). After treatment, LPO levels remained higher in birds that had clinical disease (infected group), whereas the activity of SOD and GPx enzymes did not differ between groups. Activity levels of creatine kinase (CK) and pyruvate kinase (PK) were higher in the group of chickens with clinical disease before treatment. Post-treatment, no differences were observed between groups in terms of CK; however, PK activity remained high in these animals. In the hens that died, there were lesions characteristic of avian colibacillosis, with ovary involvement, explaining the low laying activity of the birds at their peak of production. For 10 days after starting treatment, the percentage of laying increased to 90%. Therefore, we conclude that colibacillosis interferes with the phosphotransfer network by stimulating ATP production, in addition to causing oxidative stress of the birds during laying, that negatively affects health and productive efficiency.


Assuntos
Diarreia/veterinária , Infecções por Escherichia coli/veterinária , Escherichia coli/isolamento & purificação , Ovário/microbiologia , Estresse Oxidativo , Fosfotransferases/metabolismo , Doenças das Aves Domésticas/fisiopatologia , Trifosfato de Adenosina/biossíntese , Animais , Antibacterianos/farmacologia , Galinhas , Diarreia/fisiopatologia , Metabolismo Energético , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/fisiopatologia , Fezes/microbiologia , Feminino , Testes de Sensibilidade Microbiana , Fosforilação Oxidativa , Peritônio/microbiologia
18.
EMBO J ; 38(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30850388

RESUMO

Cellular elongation requires the defined coordination of intra- and extracellular processes, but the underlying mechanisms are largely unknown. The vacuole is the biggest plant organelle, and its dimensions play a role in defining plant cell expansion rates. Here, we show that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosol in Arabidopsis thaliana We demonstrate that cell wall properties are sensed and impact on the intracellular expansion of the vacuole. Using vacuolar morphology as a quantitative read-out for intracellular growth processes, we reveal that the underlying cell wall sensing mechanism requires interaction of extracellular leucine-rich repeat extensins (LRXs) with the receptor-like kinase FERONIA (FER). Our data suggest that LRXs link plasma membrane-localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the cell. This mechanism coordinates the onset of cell wall acidification and loosening with the increase in vacuolar size.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Fosfotransferases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas/metabolismo , Vacúolos/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal
19.
Plant Sci ; 280: 348-354, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824014

RESUMO

This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.


Assuntos
Citrus sinensis/enzimologia , Fosfofrutoquinases/metabolismo , Fosfotransferases/metabolismo , Citrus sinensis/genética , Clonagem Molecular , Difosfatos/metabolismo , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Expressão Gênica , Cinética , Complexos Multiproteicos , Fosfofrutoquinases/genética , Fosforilação , Fosfotransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes
20.
Cell Host Microbe ; 25(3): 454-462.e6, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30827827

RESUMO

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.


Assuntos
Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/enzimologia , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Células Eucarióticas/microbiologia , Doença dos Legionários/microbiologia , Fosforilação , Fatores de Virulência/metabolismo
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