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1.
PLoS Comput Biol ; 16(8): e1007966, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760072

RESUMO

Protein activity is often regulated by ligand binding or by post-translational modifications such as phosphorylation. Moreover, proteins that are regulated in this way often contain multiple ligand binding sites or modification sites, which can operate to create an ultrasensitive dose response. Here, we consider the contribution of the individual modification/binding sites to the activation process, and how their individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable conformational free energy contributions from distinct sites, and associate a so-called activation parameter to each site. Our analysis shows that the ultrasensitivity generally increases as the conformational free energy contribution from one or more sites is strengthened. Furthermore, ultrasensitivity depends on the mean of the activation parameters and not on their variability. In some cases, we find that the best way to maximize ultrasensitivity is to make the contribution from all sites as strong as possible. These results provide insights into the performance objectives of multiple modification/binding sites and thus help gain a greater understanding of signaling and its role in diseases.


Assuntos
Sítios de Ligação/fisiologia , Metabolismo Energético/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas , Transdução de Sinais/fisiologia , Ligantes , Modelos Biológicos , Fosforilação/fisiologia , Conformação Proteica , Subunidades Proteicas , Proteínas/química , Proteínas/metabolismo , Termodinâmica
2.
PLoS Pathog ; 16(8): e1008745, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841296

RESUMO

Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/biossíntese , Multimerização Proteica , Proteínas Tirosina Fosfatases/metabolismo , Vibrio cholerae/fisiologia , Proteínas de Bactérias/genética , Fosforilação/fisiologia , Polissacarídeos Bacterianos/genética , Proteínas Tirosina Fosfatases/genética
4.
Am J Physiol Renal Physiol ; 319(2): F284-F291, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32686524

RESUMO

Podocyte dysfunction contributes to proteinuric chronic kidney disease. A number of key proteins are essential for podocyte function, including nephrin, podocin, CD2-associated protein (CD2AP), synaptopodin, and α-actinin-4 (ACTN4). Although most of these proteins were first identified through genetic studies associated with human kidney disease, subsequent studies have identified phosphorylation of these proteins as an important posttranslational event that regulates their function. In this review, a brief overview of the function of these key podocyte proteins is provided. Second, the role of phosphorylation in regulating the function of these proteins is described. Third, the association between these phosphorylation pathways and kidney disease is reviewed. Finally, challenges and future directions in studying phosphorylation are discussed. Better characterization of these phosphorylation pathways and others yet to be discovered holds promise for translating this knowledge into new therapies for patients with proteinuric chronic kidney disease.


Assuntos
Glomérulos Renais/metabolismo , Proteínas de Membrana/metabolismo , Fosforilação/fisiologia , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Humanos , Nefropatias/metabolismo
5.
Nat Commun ; 11(1): 3146, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561718

RESUMO

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Biossíntese de Proteínas/fisiologia , Capuzes de RNA/metabolismo , Biologia Computacional , Fator de Iniciação 4E em Eucariotos/genética , Proteínas Intrinsicamente Desordenadas/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/fisiologia
6.
Proc Natl Acad Sci U S A ; 117(23): 13127-13137, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434921

RESUMO

Stomatal guard cells control gas exchange that allows plant photosynthesis but limits water loss from plants to the environment. In Arabidopsis, stomatal development is mainly controlled by a signaling pathway comprising peptide ligands, membrane receptors, a mitogen-activated protein kinase (MAPK) cascade, and a set of transcription factors. The initiation of the stomatal lineage requires the activity of the bHLH transcription factor SPEECHLESS (SPCH) with its partners. Multiple kinases were found to regulate SPCH protein stability and function through phosphorylation, yet no antagonistic protein phosphatase activities have been identified. Here, we identify the conserved PP2A phosphatases as positive regulators of Arabidopsis stomatal development. We show that mutations in genes encoding PP2A subunits result in lowered stomatal production in Arabidopsis Genetic analyses place the PP2A function upstream of SPCH. Pharmacological treatments support a role for PP2A in promoting SPCH protein stability. We further find that SPCH directly binds to the PP2A-A subunits in vitro. In plants, nonphosphorylatable SPCH proteins are less affected by PP2A activity levels. Thus, our research suggests that PP2A may function to regulate the phosphorylation status of the master transcription factor SPCH in stomatal development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Estômatos de Plantas/crescimento & desenvolvimento , Proteína Fosfatase 2/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação , Fosforilação/fisiologia , Estômatos de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/isolamento & purificação , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tabaco/genética
7.
Life Sci ; 255: 117834, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454158

RESUMO

AIMS: Hydrogen sulfide (H2S) is shown in ocular tissues and suggested to involve in the regulation of retinal circulation. However, the mechanism of H2S-induced relaxation on retinal artery is not clarified yet. Herein, we aimed to evaluate the role of several calcium (Ca2+) signaling and Ca2+ sensitization mechanisms in the relaxing effect of H2S donor, NaHS, on retinal arteries. MATERIALS AND METHODS: Relaxing effects of NaHS (10-5-3 × 10-3M) were determined on precontracted retinal arteries in Ca2+ free medium as well as in the presence of the inhibitors of Ca2+ signaling and Ca2+ sensitization mechanisms. Additively, Ca2+ sensitivity of the contractile apparatus were evaluated by CaCl2-induced contractions in the presence of NaHS (3 × 10-3M). Functional experiments were furtherly assessed by protein and/or mRNA expressions, as appropriate. KEY FINDINGS: The relaxations to NaHS were preserved in Ca2+ free medium while NaHS pretreatment decreased the responsiveness to CaCl2. The inhibitors of plasmalemmal Ca2+-ATPase, sarcoplasmic-endoplasmic reticulum Ca2+-ATPase, Na+-Ca2+ ion-exchanger and myosin light chain kinase (MLCK) unchanged the relaxations to NaHS. Likewise, Ca2+ sensitization mechanisms including, rho kinase, protein kinase C and tyrosine kinase were unlikely to mediate the relaxation to NaHS in retinal artery. Whereas, a marked reduction was determined in NaHS-induced relaxations in the presence of MLCP inhibitor, calyculin A. Supportively, NaHS pretreatment significantly reduced phosphorylation of MYPT1-subunit of MLCP. SIGNIFICANCE: The relaxing effect of NaHS in retinal artery is likely to be related to the activation of MLCP and partly, to decrement in Ca2+ sensitivity of contractile apparatus.


Assuntos
Cálcio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Artéria Retiniana/metabolismo , Animais , Cloreto de Cálcio/administração & dosagem , Sinalização do Cálcio/fisiologia , Bovinos , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Fosforilação/fisiologia , Sulfetos/administração & dosagem , Sulfetos/farmacologia
8.
Proc Natl Acad Sci U S A ; 117(22): 11865-11874, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32444484

RESUMO

Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing-proposed in vertebrate muscle-is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca2+-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.


Assuntos
Músculo Estriado/fisiologia , Miosinas/metabolismo , Fosforilação/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Artrópodes/fisiologia , Evolução Molecular , Invertebrados/fisiologia , Modelos Moleculares , Contração Muscular , Relaxamento Muscular , Miosinas/química , Estrutura Secundária de Proteína , Aranhas/fisiologia , Vertebrados/fisiologia
9.
Metabolism ; 108: 154257, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32370945

RESUMO

BACKGROUND: Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. METHODS: To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. RESULTS: Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. CONCLUSIONS: Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.


Assuntos
AMP Desaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Proteólise , Ubiquitina/metabolismo
10.
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
11.
Invest Ophthalmol Vis Sci ; 61(5): 10, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32396631

RESUMO

Purpose: A burst in phagocytosis of spent photoreceptor outer fragments by RPE is a rhythmic process occurring 1 to 2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE. Methods: Here, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R. Results: D2R knockout (KO) mice do not show a daily burst of phagocytic activity after the onset of light. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT 23 and ZT 1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. Consistent with the gene expression data, phosphorylation of focal adhesion kinase (FAK) did not increase significantly in KO mice at ZT 1. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between wild-type (WT) and D2R KO mice at the age of 3 and 12 months. Conclusions: Our data suggest that removal of D2R prevents the burst of phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points toward a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence of the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to 1 year of age.


Assuntos
Fagocitose , Receptores de Dopamina D2/fisiologia , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/fisiologia , Animais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/patologia , Fosforilação/fisiologia , Tomografia de Coerência Óptica , Regulação para Cima/fisiologia
12.
PLoS One ; 15(4): e0228121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236094

RESUMO

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and ß-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.


Assuntos
Transdução de Sinal Luminoso/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Opsinas de Bastonetes/metabolismo , beta-Arrestina 1/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosforilação/fisiologia , Ligação Proteica , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes de Fusão/genética , Opsinas de Bastonetes/química , Opsinas de Bastonetes/genética , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , beta-Arrestina 1/química
13.
PLoS One ; 15(4): e0230814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251425

RESUMO

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. ßPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. ßPix is expressed as alternatively spliced variants, including the ubiquitous isoform ßPix-a and the neuronal isoforms ßPix-b and ßPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of ßPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from ßPix neuronal isoform knockout (ßPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating ßPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal ßPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal ßPix isoforms may promote microtubule stabilization during neurite development. ßPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either ßPix neuronal isoform in the ßPix-NIKO neurons restores Stathmin1 phosphorylation levels, with ßPix-d having a greater effect than ßPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via ßPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that ßPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal ßPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.


Assuntos
Crescimento Neuronal/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/fisiologia , Estatmina/metabolismo , Tubulina (Proteína)/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Acetilação , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Neuritos/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação/fisiologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia
14.
Nat Commun ; 11(1): 1838, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296066

RESUMO

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.


Assuntos
Proteínas de Arabidopsis/metabolismo , NADPH Oxidases/metabolismo , Imunidade Vegetal/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , NADPH Oxidases/genética , Fosforilação/genética , Fosforilação/fisiologia , Doenças das Plantas/genética , Imunidade Vegetal/genética , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitinação/genética , Ubiquitinação/fisiologia
15.
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
16.
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
17.
Mem Inst Oswaldo Cruz ; 115: e190357, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130369

RESUMO

BACKGROUND: Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3ß, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3ß during Dengue virus-2 (DENV-2) infection was investigated. METHODS: GSK3ß participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS: Phosphorylation of GSK3ß-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3ß reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3ß. CONCLUSIONS: The results suggest that GSK3ß participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.


Assuntos
Vírus da Dengue/enzimologia , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/fisiologia , Replicação Viral/fisiologia , Aedes/citologia , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Microscopia de Fluorescência , Fosforilação/fisiologia , Transdução de Sinais
18.
J Pharmacol Exp Ther ; 373(3): 370-380, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32205367

RESUMO

The pregnane X receptor (PXR), or nuclear receptor (NR) 1I2, is a ligand-activated NR superfamily member that is enriched in liver and intestine in mammals. Activation of PXR regulates the expression of genes encoding key proteins involved in drug metabolism, drug efflux, and drug transport. Recent mechanistic investigations reveal that post-translational modifications (PTMs), such as phosphorylation, play a critical role in modulating the bimodal function of PXR-mediated transrepression and transactivation of target gene transcription. Upon ligand binding, PXR undergoes a conformational change that promotes dissociation of histone deacetylase-containing multiprotein corepressor protein complexes while simultaneously favoring recruitment histone acetyl transferase-containing complexes. Here we describe a novel adenoviral vector used to deliver and recover recombinant human PXR protein from primary cultures of hepatocytes. Using liquid chromatography and tandem mass spectrometry we report here that PXR is phosphorylated at amino acid residues threonine 135 (T135) and serine 221 (S221). Biochemical analysis reveals that these two residues play an important regulatory role in the cycling of corepressor and coactivator multiprotein complexes. These data further our foundational knowledge regarding the specific role of PTMs, namely phosphorylation, in regulating the biology of PXR. Future efforts are focused on using the novel tools described here to identify additional PTMs and protein partners of PXR in primary cultures of hepatocytes, an important experimental model system. SIGNIFICANCE STATEMENT: Pregnane X receptor (PXR), or nuclear receptor 1I2, is a key master regulator of drug-inducible CYP gene expression in liver and intestine in mammals. The novel biochemical tools described in this study demonstrate for the first time that in cultures of primary hepatocytes, human PXR is phosphorylated at amino acid residues threonine 135 (T135) and serine 221 (S221). Moreover, phosphorylation of PXR promotes the transrepression of its prototypical target gene CYP3A4 through modulating its interactions with coregulatory proteins.


Assuntos
Fosforilação/fisiologia , Receptor de Pregnano X/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Camundongos , Processamento de Proteína Pós-Traducional/fisiologia , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Treonina/metabolismo
19.
PLoS One ; 15(2): e0229036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084172

RESUMO

LAT molecules defective in ubiquitination have an increased half-life and induce enhanced signaling when expressed in T cells. In this study, we have examined the role of ubiquitination in regulating LAT endocytosis, recycling, and degradation in resting and stimulated T cells. By tracking and comparing plasma membrane-labeled wild type and ubiquitination-resistant 2KR LAT, we find that ubiquitination promotes the degradation of surface LAT in T cells. Activation of T cells increases LAT ubiquitination and promotes trafficking of internalized LAT to lysosomes for degradation. Ubiquitination of LAT does not change internalization rates from the cell surface, but prevents efficient recycling of LAT to the surface of T cells. Our study demonstrates that surface LAT levels are tightly controlled by ubiquitination. LAT in unstimulated cells lacks ubiquitin allowing for increased LAT stability and efficient T cell activation upon TCR triggering; ubiquitination leads to efficient removal of LAT after activation.


Assuntos
Ativação Linfocitária/fisiologia , Ubiquitinação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Endocitose/fisiologia , Humanos , Immunoblotting , Lisossomos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Transdução de Sinais/fisiologia
20.
Biochem Soc Trans ; 48(1): 199-205, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32065230

RESUMO

Tyrosine kinases are signaling molecules that are common to all metazoans and are involved in the regulation of many cellular processes such as proliferation and survival. While most attention has been devoted to tyrosine kinases signaling at the plasma membrane and the cytosol, very little attention has been dedicated to signaling at endomembranes. In this review, I will discuss recent evidence that we obtained on signaling of tyrosine kinases at the surface of the endoplasmic reticulum (ER), as well as in the lumen of this organelle. I will discuss how tyrosine kinase signaling might regulate ER proteostasis and the implication thereof to general cell physiology.


Assuntos
Retículo Endoplasmático/enzimologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Citosol/enzimologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Fosforilação/fisiologia , Proteostase/fisiologia , Resposta a Proteínas não Dobradas
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