Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 35
Filtrar
1.
Cell ; 159(3): 623-34, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25417112

RESUMO

S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of over a thousand S-nitrosylated proteins, few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by iNOS-S100A8/A9 were revealed by proteomic analysis. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS-S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent posttranslational modification of selected targets by primary sequence motif recognition.


Assuntos
Complexos Multiproteicos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Interferon gama/metabolismo , Lipoproteínas LDL/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência
2.
Nat Immunol ; 17(11): 1252-1262, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27595231

RESUMO

The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/-) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno/imunologia , Viroses/imunologia , Viroses/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Imunidade Inata , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Fosforilação , Ligação Proteica , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/virologia , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Ubiquitinação , Viroses/virologia , Replicação Viral
3.
Cell ; 149(1): 88-100, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22386318

RESUMO

Posttranscriptional regulatory mechanisms superimpose "fine-tuning" control upon "on-off" switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. The GAIT (gamma-interferon-activated inhibitor of translation) complex repressed VEGF-A synthesis to a low, constant rate independent of VEGF-A mRNA expression levels. Dynamic model simulations predicted an inhibitory GAIT-element-interacting factor to account for this relationship and led to the identification of a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), a GAIT constituent that mediates binding to target transcripts. The truncated protein, EPRS(N1), shields GAIT-element-bearing transcripts from the inhibitory GAIT complex, thereby dictating a "translational trickle" of GAIT target proteins. EPRS(N1) mRNA is generated by polyadenylation-directed conversion of a Tyr codon in the EPRS-coding sequence to a stop codon (PAY(∗)). Genome-wide analysis revealed multiple candidate PAY(∗) targets, including the authenticated target RRM1, suggesting a general mechanism for production of C terminus-truncated regulatory proteins.


Assuntos
Aminoacil-tRNA Sintetases/genética , Regulação da Expressão Gênica , Genoma Humano , Biossíntese de Proteínas , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Códon de Terminação , Humanos , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Poliadenilação , Transcriptoma , Células U937 , Fator A de Crescimento do Endotélio Vascular/genética
4.
Mol Cell ; 73(3): 446-457.e6, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30612880

RESUMO

Multisite phosphorylation of kinases can induce on-off or graded regulation of catalytic activity; however, its influence on substrate specificity remains unclear. Here, we show that multisite phosphorylation of ribosomal protein S6 kinase 1 (S6K1) alters target selection. Agonist-inducible phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) by S6K1 in monocytes and adipocytes requires not only canonical phosphorylation at Thr389 by mTORC1 but also phosphorylation at Ser424 and Ser429 in the C terminus by cyclin-dependent kinase 5 (Cdk5). S6K1 phosphorylation at these additional sites induces a conformational switch and is essential for high-affinity binding and phosphorylation of EPRS, but not canonical S6K1 targets, e.g., ribosomal protein S6. Unbiased proteomic analysis identified additional targets phosphorylated by multisite phosphorylated S6K1 in insulin-stimulated adipocytes-namely, coenzyme A synthase, lipocalin 2, and cortactin. Thus, embedded within S6K1 is a target-selective kinase phospho-code that integrates signals from mTORC1 and Cdk5 to direct an insulin-stimulated, post-translational metabolon determining adipocyte lipid metabolism.


Assuntos
Adipócitos/enzimologia , Metabolismo dos Lipídeos , Células Mieloides/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Aminoacil-tRNA Sintetases/metabolismo , Animais , Quinase 5 Dependente de Ciclina/metabolismo , Ativação Enzimática , Células HEK293 , Células Hep G2 , Humanos , Insulina/farmacologia , Interferon gama/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Células Mieloides/efeitos dos fármacos , Fosforilação , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Especificidade por Substrato , Células U937
6.
Nature ; 542(7641): 357-361, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28178239

RESUMO

Metabolic pathways that contribute to adiposity and ageing are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting that there are additional downstream effectors of this pathway. Here we identify glutamyl-prolyl-tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to adiposity and ageing. Phosphorylation of EPRS at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex, which is required for execution of noncanonical functions of EPRS beyond protein synthesis. To investigate the physiological function of EPRS phosphorylation, we generated Eprs knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, similar to S6K1-deficient mice and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent. Substitution of the EprsS999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating that EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the multisynthetase complex. Interaction screening revealed that phospho-EPRS binds SLC27A1 (that is, fatty acid transport protein 1, FATP1), inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors that are critical for metabolic phenotypes.


Assuntos
Adiposidade , Aminoacil-tRNA Sintetases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adipócitos/metabolismo , Envelhecimento/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Animais , Peso Corporal , Membrana Celular/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Insulina/metabolismo , Longevidade/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Mutação , Tamanho do Órgão , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Transporte Proteico , Proteína Regulatória Associada a mTOR , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência
7.
Mol Cell ; 47(4): 656-63, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22771119

RESUMO

Multiple eukaryotic ribosomal proteins (RPs) are co-opted for extraribosomal "moonlighting" activities, but paradoxically, RPs exhibit rapid turnover when not ribosome-bound. In one illustrative case of a functional extraribosomal RP, interferon (IFN)-γ induces ribosome release of L13a and assembly into the IFN-gamma-activated inhibitor of translation (GAIT) complex for translational control of a subset of inflammation-related proteins. Here we show GAPDH functions as a chaperone, shielding newly released L13a from proteasomal degradation. However, GAPDH protective activity is lost following cell treatment with oxidatively modified low density lipoprotein and IFN-γ. These agonists stimulate S-nitrosylation at Cys(247) of GAPDH, which fails to interact with L13a, causing proteasomal degradation of essentially the entire cell complement of L13a and defective translational control. Evolution of extraribosomal RP activities might require coevolution of protective chaperones, and pathological disruption of either protein, or their interaction, presents an alternative mechanism of diseases due to RP defects, and targets for therapeutic intervention.


Assuntos
Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Marcha/genética , Inativação Gênica , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Células Mieloides/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Células U937 , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
J Biol Chem ; 293(23): 8843-8860, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29643180

RESUMO

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH-S-transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Caspases/metabolismo , Aminoacil-tRNA Sintetases/química , Domínio Catalítico , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteólise
9.
Methods ; 113: 72-82, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27729295

RESUMO

Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized for decades, but the contribution of post-translational modification to their primary role in tRNA charging and decryption of genetic code remains unclear. In contrast, phosphorylation is essential for performance of diverse noncanonical functions of AARSs unrelated to protein synthesis. Phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our laboratory for more than a decade, and has served as an archetype for studies of other AARSs. EPRS is a constituent of the IFN-γ-activated inhibitor of translation (GAIT) complex that directs transcript-selective translational control in myeloid cells. Stimulus-dependent phosphorylation of EPRS is essential for its release from the parental multi-aminoacyl tRNA synthetase complex (MSC), for binding to other GAIT complex proteins, and for regulating the binding to target mRNAs. Importantly, phosphorylation is the common driving force for the context- and stimulus-dependent release, and non-canonical activity, of other AARSs residing in the MSC, for example, lysyl tRNA synthetase (KARS). Here, we describe the concepts and experimental methodologies we have used to investigate the influence of phosphorylation on the structure and function of EPRS. We suggest that application of these approaches will help to identify new functional phosphorylation event(s) in other AARSs and elucidate their possible roles in noncanonical activities.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Bioensaio , Monócitos/metabolismo , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , RNA de Transferência de Prolina/metabolismo , Aminoacil-tRNA Sintetases/genética , Animais , Anticorpos/química , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Monócitos/citologia , Radioisótopos de Fósforo , Fosforilação , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência de Prolina/genética
10.
Mol Cell ; 35(2): 164-80, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19647514

RESUMO

Glutamyl-prolyl tRNA synthetase (EPRS) is a component of the heterotetrameric gamma-interferon-activated inhibitor of translation (GAIT) complex that binds 3'UTR GAIT elements in multiple interferon-gamma (IFN-gamma)-inducible mRNAs and suppresses their translation. Here, we elucidate the specific EPRS phosphorylation events that regulate GAIT-mediated gene silencing. IFN-gamma induces sequential phosphorylation of Ser(886) and Ser(999) in the noncatalytic linker connecting the synthetase cores. Phosphorylation of both sites is essential for EPRS release from the parent tRNA multisynthetase complex. Ser(886) phosphorylation is required for the interaction of NSAP1, which blocks EPRS binding to target mRNAs. The same phosphorylation event induces subsequent binding of ribosomal protein L13a and GAPDH and restores mRNA binding. Finally, Ser(999) phosphorylation directs the formation of a functional GAIT complex that binds initiation factor eIF4G and represses translation. Thus, two-site phosphorylation provides structural and functional pliability to EPRS and choreographs the repertoire of activities that regulates inflammatory gene expression.


Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Inativação Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Células Cultivadas , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Interferon gama/metabolismo , Interferon gama/fisiologia , Modelos Genéticos , Fosforilação , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , RNA Mensageiro/fisiologia , Proteínas Ribossômicas/metabolismo , Serina/metabolismo
11.
Sensors (Basel) ; 17(9)2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28869538

RESUMO

Carbon fiber-based materials possess excellent mechanical properties and show linear piezoresistive behavior, which make them good candidate materials for strain measurements. They have the potential to be used as sensors for various applications such as damage detection, stress analysis and monitoring of manufacturing processes and quality. In this paper, carbon fiber sensors are prepared to perform reliable strain measurements. Both experimental and computational studies were carried out on commercially available carbon fibers in order to understand the response of the carbon fiber sensors due to changes in the axial strain. Effects of parameters such as diameter, length, and epoxy-hardener ratio are discussed. The developed numerical model was calibrated using laboratory-based experimental data. The results of the current study show that sensors with shorter lengths have relatively better sensitivity. This is due to the fact short fibers have low initial resistance, which will increase the change of resistance over initial resistance. Carbon fibers with low number of filaments exhibit linear behavior while nonlinear behavior due to transverse resistance is significant in fibers with large number of filaments. This study will allow researchers to predict the behavior of the carbon fiber sensor in real life and it will serve as a basis for designing carbon fiber sensors to be used in different applications.

12.
Mol Cell ; 29(6): 679-90, 2008 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-18374644

RESUMO

The heterotetrameric GAIT complex suppresses translation of selected mRNAs in interferon-gamma-activated monocytic cells. Specificity is dictated by glutamyl-prolyl tRNA synthetase (EPRS) binding to a 3'UTR element in target mRNAs. EPRS consists of two synthetase cores joined by a linker containing three WHEP domains of unknown function. Here we show the critical role of EPRS WHEP domains in targeting and regulating GAIT complex binding to RNA. The upstream WHEP pair directs high-affinity binding to GAIT element-bearing mRNAs, while the overlapping, downstream pair binds NSAP1, which inhibits mRNA binding. Interaction of EPRS with ribosomal protein L13a and GAPDH induces a conformational switch that rescues mRNA binding and restores translational control. Total reconstitution from purified components indicates that the four GAIT proteins are necessary and sufficient for self-assembly of a functional complex. Our results establish the essentiality of WHEP domains in the noncanonical function of EPRS in regulating inflammatory gene expression.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Regiões 3' não Traduzidas/genética , Aminoacil-tRNA Sintetases/genética , Sítios de Ligação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Inflamação/enzimologia , Cinética , Substâncias Macromoleculares/metabolismo , Monócitos/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Ribossômica L3 , Proteínas Ribossômicas/metabolismo , Transcrição Gênica
13.
Mol Cell ; 32(3): 371-82, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18995835

RESUMO

Phosphorylation of ribosomal protein L13a is essential for translational repression of inflammatory genes by the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex. Here we show that IFN-gamma activates a kinase cascade in which death-associated protein kinase-1 (DAPK) activates zipper-interacting protein kinase (ZIPK), culminating in L13a phosphorylation on Ser(77), L13a release from the ribosome, and translational silencing of GAIT element-bearing target mRNAs. Remarkably, both kinase mRNAs contain functional 3'UTR GAIT elements, and thus the same inhibitory pathway activated by the kinases is co-opted to suppress their expression. Inhibition of DAPK and ZIPK facilitates cell restoration to the basal state and allows renewed induction of GAIT target transcripts by repeated stimulation. Thus, the DAPK-ZIPK-L13a axis forms a unique regulatory module that first represses, then repermits inflammatory gene expression. We propose that the module presents an important checkpoint in the macrophage "resolution of inflammation" program, and that pathway defects may contribute to chronic inflammatory disorders.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Inflamação/genética , MAP Quinase Quinase Quinases/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , Inflamação/enzimologia , Inflamação/fisiopatologia , Fragmentos de Peptídeos/química , Fosforilação , Plasmídeos , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Células U937
14.
Proc Natl Acad Sci U S A ; 108(4): 1415-20, 2011 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-21220307

RESUMO

Cyclin-dependent kinase 5 (Cdk5) is an atypical but essential member of the Cdk kinase family, and its dysregulation or deletion has been implicated in inflammation-related disorders by an undefined mechanism. Here we show that Cdk5 is an indispensable activator of the GAIT (IFN-γ-activated inhibitor of translation) pathway, which suppresses expression of a posttranscriptional regulon of proinflammatory genes in myeloid cells. Through induction of its regulatory protein, Cdk5R1 (p35), IFN-γ activates Cdk5 to phosphorylate Ser(886) in the linker domain of glutamyl-prolyl tRNA synthetase (EPRS), the initial event in assembly of the GAIT complex. Cdk5/p35 also induces, albeit indirectly via a distinct kinase, phosphorylation of Ser(999), the second essential event in GAIT pathway activation. Diphosphorylated EPRS is released from its residence in the tRNA multisynthetase complex for immediate binding to NS1-associated protein and subsequent binding to ribosomal protein L13a and GAPDH. The mature heterotetrameric GAIT complex binds the 3' UTR GAIT element of VEGF-A and other target mRNAs and suppresses their translation in myeloid cells. Inhibition of Cdk5/p35 inhibits both EPRS phosphorylation events, prevents EPRS release from the tRNA multisynthetase complex, and blocks translational suppression of GAIT element-bearing mRNAs, resulting in increased expression of inflammatory proteins. Our study reveals a unique role of Cdk5/p35 in activation of the major noncanonical function of EPRS, namely translational control of macrophage inflammatory gene expression.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas/genética , Serina/metabolismo , Transcrição Gênica/genética , Aminoacil-tRNA Sintetases/genética , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Humanos , Immunoblotting , Interferon gama/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/metabolismo , Serina/genética , Transcrição Gênica/efeitos dos fármacos , Células U937 , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Trends Biochem Sci ; 34(7): 324-31, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19535251

RESUMO

Functionally related genes are coregulated by specific RNA-protein interactions that direct transcript-selective translational control. In myeloid cells, interferon (IFN)-gamma induces formation of the heterotetrameric, IFN-gamma-activated inhibitor of translation (GAIT) complex comprising glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This complex binds defined 3' untranslated region elements within a family of inflammatory mRNAs and suppresses their translation. IFN-gamma-dependent phosphorylation, and consequent release of EPRS and L13a from the tRNA multisynthetase complex and 60S ribosomal subunit, respectively, regulates GAIT complex assembly. EPRS recognizes and binds target mRNAs, NSAP1 negatively regulates RNA binding, and L13a inhibits translation initiation by binding eukaryotic initiation factor 4G. Repression of a post-transcriptional regulon by the GAIT system might contribute to the resolution of chronic inflammation.


Assuntos
Inflamação/genética , Interferon gama/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Fator de Iniciação Eucariótico 4G/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , MicroRNAs/metabolismo , Células Mieloides/metabolismo , Proteínas Ribossômicas/metabolismo
16.
Trends Biochem Sci ; 32(4): 158-64, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17321138

RESUMO

Multi-component, macromolecular complexes perform essential cellular functions that require spatial or temporal coordination of activities. Complexes also facilitate co-regulation of protein amounts and cellular localization of individual components. We propose a novel function of multi-component complexes as depots for regulatory proteins that, upon release, acquire new auxiliary functions. We further propose that component release is inducible and context-dependent. We describe two cases in which multi-component assemblies - the ribosome and tRNA multi-synthetase complex--function as depots. Both complexes have crucial roles in supporting protein synthesis but they also release regulatory proteins for inflammation-responsive, transcript-specific translational control. Recent evidence indicates that other macromolecular assemblies might be sources for proteins with auxiliary functions, and the depot mechanism might be widespread in nature.


Assuntos
Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , Animais , Humanos , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica , Biossíntese de Proteínas , Proteínas/química , Ribossomos/metabolismo
17.
Heliyon ; 8(8): e10063, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35991970

RESUMO

This work presents a hybrid thermography, computational, and Artificial Neural Networks (ANN) approach to characterize beneath the surface defects in composites. Computational simulations are created to model thermography experiments carried out on composite plates with controlled damage in the form of drilled holes. The computational models are then extended to create hypothetical composite component geometries of plates and pipes with embedded defects of varying sizes and shapes. The data from the computational simulations are fed to artificial neural networks to train them to predict and characterize defect sizes and shapes. The predictions from the neural networks are compared to the actual dimensions from the computational models. These predictions show a high level of accuracy especially when quantifying thermal image information and using it to train the neural network. This accuracy is around 10% and 19% for predicting defect depth in plates and pipes, respectively. This hybrid approach has the advantage of not relying on experimental data (experiments were used only for validation) and predicting damage shape and size. This suggests that the methodology used in this study combining lock-in thermography experiments, computational simulations, and ANNs is a viable method for a potential nondestructive testing (NDT) method for detecting embedded defects within composite pipes in real applications. What makes this approach attractive is that it can be used with live thermal images that can be fed directly into the ANN model.

18.
Sci Rep ; 10(1): 20297, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219307

RESUMO

Fibroblast-like synoviocytes (FLS) play a critical role in the pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment. We analyzed high throughput gene expression and chromatin accessibility data from human and mouse FLS from our and other studies available on public repositories, with the goal of identifying the persistently reprogrammed signaling pathways driven by chronic inflammation. We found that the gene expression changes induced by short-term tumor necrosis factor-alpha (TNF) treatment were largely sustained in the FLS exposed to chronic inflammation. These changes that included both activation and repression of gene expression, were accompanied by the remodeling of chromatin accessibility. The sustained activated genes (SAGs) included established pro-inflammatory signaling components known to act at multiple levels of NF-kappaB, STAT and AP-1 signaling cascades. Interestingly, the sustained repressed genes (SRGs) included critical mediators and targets of the BMP signaling pathway. We thus identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation. We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence.


Assuntos
Artrite Reumatoide/imunologia , Transdução de Sinais/imunologia , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Reumatoide/patologia , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Conjuntos de Dados como Assunto , Epigênese Genética/imunologia , Fibroblastos , Humanos , Camundongos , Cultura Primária de Células , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Membrana Sinovial/imunologia , Sinoviócitos/imunologia , Sinoviócitos/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/genética
19.
Sci Rep ; 10(1): 5304, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210302

RESUMO

A computational design methodology is reported to propose a high-performance composite for backside encapsulation of concentrated photovoltaic (CPV) systems for enhanced module life and electrical power. Initially, potential polymer composite systems that are expected to provide the target properties, such as thermal conductivity, coefficient of thermal expansion, and long-term shear modulus are proposed using in-house built design codes. These codes are based on differential effective medium theory and mean-field homogenization, which lead to the selection of matrix, filler, volume fractions, and type of particulates. Thermoplastic polyurethane (TPU) loaded with ceramics fillers of a minimum spherical diameter of 6 µm are found potential composites. Some representative samples are synthesized through the melt-mixing and compression-molding route and characterized. The target properties including thermal conductivity, coefficient of thermal expansion, viscoelastic parameters, and long-term shear modulus are measured and used to evaluate the performance of CPV modules using previously published finite element model. The proposed composite can drag the cell temperature down by 5.8 °C when compared with neat TPU which leads to a 4.3% increase in electrical power along with a reasonable module life. It is expected that this approach will make a baseline for the effective production of polymer composites in various industrial applications.

20.
Ann N Y Acad Sci ; 1463(1): 45-59, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31919867

RESUMO

Bone remodeling is achieved through the coupled activities of osteoclasts and osteoblasts that are controlled by many locally generated secreted factors, including WNT5A. While previous studies have demonstrated that osteoblast-derived WNT5A promotes osteoclastogenesis, the function of osteoclast-derived WNT5A on bone remodeling has remained unexplored. We examined the effects of osteoclast-derived WNT5A on bone homeostasis by utilizing the Cathepsin K-Cre (Ctsk-Cre) mouse to conditionally delete Wnt5a in mature osteoclasts. These mice exhibited reduced trabecular and cortical bone. The low bone-mass phenotype was driven by decreased bone formation, not osteoclast-mediated bone resorption, as osteoclast number and serum CTX marker were unchanged. Furthermore, molecular analysis of osteoclast- and osteoblast-derived WNT5A identified a serine-phosphorylated WNT5A that is unique to RANKL-treated macrophages mimicking osteoclasts. This study suggests a new paradigm in which WNT5A has opposing effects on bone remodeling that are dependent on the cell of origin, an effect that may result from cell type-specific differential posttranslational modifications of WNT5A.


Assuntos
Reabsorção Óssea/metabolismo , Deleção de Genes , Osteoclastos/metabolismo , Osteogênese/fisiologia , Proteína Wnt-5a/deficiência , Animais , Reabsorção Óssea/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células RAW 264.7 , Proteína Wnt-5a/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA