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1.
Nat Commun ; 10(1): 3396, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363119

RESUMO

Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.

2.
Nature ; 567(7746): 109-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787439

RESUMO

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.


Assuntos
Quirópteros/virologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Especificidade de Hospedeiro , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Zoonoses/imunologia , Zoonoses/virologia , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Galinhas/genética , Galinhas/imunologia , Quirópteros/genética , Quirópteros/imunologia , Quirópteros/metabolismo , Feminino , Perfilação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/imunologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Sistema Respiratório/virologia , Suínos/genética , Suínos/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Replicação Viral , Zoonoses/genética , Zoonoses/metabolismo
3.
Science ; 361(6404): 810-813, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30026316

RESUMO

RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.


Assuntos
Artrite/genética , Doenças Inflamatórias Intestinais/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Imunodeficiência Combinada Severa/genética , Alelos , Artrite/imunologia , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Linfopenia/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linhagem , Imunodeficiência Combinada Severa/imunologia
4.
Nat Commun ; 7: 13992, 2016 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-28008925

RESUMO

Mutations in genes encoding components of the immune system cause primary immunodeficiencies. Here, we study a patient with recurrent atypical mycobacterial infection and early-onset metastatic bladder carcinoma. Exome sequencing identified two homozygous missense germline mutations, P733L and P832S, in the JAK1 protein that mediates signalling from multiple cytokine receptors. Cells from this patient exhibit reduced JAK1 and STAT phosphorylation following cytokine stimulations, reduced induction of expression of interferon-regulated genes and dysregulated cytokine production; which are indicative of signalling defects in multiple immune response pathways including Interferon-γ production. Reconstitution experiments in the JAK1-deficient cells demonstrate that the impaired JAK1 function is mainly attributable to the effect of the P733L mutation. Further analyses of the mutant protein reveal a phosphorylation-independent role of JAK1 in signal transduction. These findings clarify JAK1 signalling mechanisms and demonstrate a critical function of JAK1 in protection against mycobacterial infection and possibly the immunological surveillance of cancer.


Assuntos
Alelos , Janus Quinase 1/genética , Mutação/genética , Infecções por Mycobacterium/enzimologia , Infecções por Mycobacterium/genética , Sequência de Aminoácidos , Sequência de Bases , Células Sanguíneas/metabolismo , Criança , Pré-Escolar , Citocinas/sangue , Suscetibilidade a Doenças , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Janus Quinase 1/química , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , TYK2 Quinase/metabolismo , Adulto Jovem
5.
Endocrinology ; 157(5): 1914-28, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26982636

RESUMO

IGFs are critical for normal intrauterine and childhood growth and sustaining health throughout life. We showed previously that the production of IGF-1 and IGF-2 requires interaction with the chaperone glucose-regulated protein 94 (GRP94) and that the amount of secreted IGFs is proportional to the GRP94 activity. Therefore, we tested the hypothesis that functional polymorphisms of human GRP94 affect IGF production and thereby human health. We describe a hypomorphic variant of human GRP94, P300L, whose heterozygous carriers have 9% lower circulating IGF-1 concentration. P300L was found first in a child with primary IGF deficiency and was later shown to be a noncommon single-nucleotide polymorphism with frequencies of 1%-4% in various populations. When tested in the grp94(-/-) cell-based complementation assay, P300L supported only approximately 58% of IGF secretion relative to wild-type GRP94. Furthermore, recombinant P300L showed impaired nucleotide binding activity. These in vitro data strongly support a causal relationship between the GRP94 variant and the decreased concentration of circulating IGF-1, as observed in human carriers of P300L. Thus, mutations in GRP94 that affect its IGF chaperone activity represent a novel causal genetic mechanism that limits IGF biosynthesis, quite a distinct mechanism from the known genes in the GH/IGF signaling network.


Assuntos
Proteínas de Choque Térmico HSP70/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Somatomedinas/biossíntese , Alelos , Análise Mutacional de DNA , Frequência do Gene , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Mutação
6.
FASEB J ; 30(2): 653-65, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26487694

RESUMO

Protein disulfide isomerase A6 (PDIA6) interacts with protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme (IRE)-1 and inhibits their unfolded protein response signaling. In this study, shRNA silencing of PDIA6 expression in insulin-producing mouse cells reduced insulin production (5-fold) and, consequently, glucose-stimulated insulin secretion (3-4-fold). This inhibition of insulin release was independent of the PDIA6-PERK interaction or PERK activity. Acute inhibition of PERK did not change the short-term response of ß cells to glucose. Rather, PDIA6 affected insulin secretion by modulating one of the activities of IRE1. At 11 mM glucose and lower, the regulated IRE1-dependent decay (RIDD) of the mRNA activity of IRE1 was activated, but not its X-box binding protein (XBP)-1 splicing activity. In the absence of PDIA6, RIDD activity toward insulin transcripts was enhanced up to 4-fold, as shown by molecular assays in cultured cells and the use of a fluorescent reporter in intact islets. Such physiologic activation of IRE1 by glucose contrasted with IRE1 activation by chemical stress, when both IRE1 activities were induced. Thus, whereas the stimulus determines the quality of IRE1 signaling, PDIA6 attenuates multiple enzymatic activities of IRE1, maintaining its signaling within a physiologically tolerable range.


Assuntos
Endorribonucleases/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/genética , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Glucose/metabolismo , Glucose/farmacologia , Humanos , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/genética , Camundongos , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Tapsigargina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
7.
J Cell Sci ; 127(Pt 17): 3649-58, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25107370

RESUMO

In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca(2+) efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER-mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Humanos
8.
Mol Biol Cell ; 25(15): 2220-34, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24899641

RESUMO

The tight coupling of protein folding pathways with disposal mechanisms promotes the efficacy of protein production in the endoplasmic reticulum (ER). It has been hypothesized that the ER-resident molecular chaperone glucose-regulated protein 94 (GRP94) is part of this quality control coupling because it supports folding of select client proteins yet also robustly associates with the lectin osteosarcoma amplified 9 (OS-9), a component involved in ER-associated degradation (ERAD). To explore this possibility, we investigated potential functions for the GRP94/OS-9 complex in ER quality control. Unexpectedly, GRP94 does not collaborate with OS-9 in ERAD of misfolded substrates, nor is the chaperone required directly for OS-9 folding. Instead, OS-9 binds preferentially to a subpopulation of GRP94 that is hyperglycosylated on cryptic N-linked glycan acceptor sites. Hyperglycosylated GRP94 forms have nonnative conformations and are less active. As a result, these species are degraded much faster than the major, monoglycosylated form of GRP94 in an OS-9-mediated, ERAD-independent, lysosomal-like mechanism. This study therefore clarifies the role of the GRP94/OS-9 complex and describes a novel pathway by which glycosylation of cryptic acceptor sites influences the function and fate of an ER-resident chaperone.


Assuntos
Lectinas/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional , Proteólise , Trifosfato de Adenosina/metabolismo , Degradação Associada com o Retículo Endoplasmático , Glicosilação , Células HEK293 , Humanos , Cinética , Lectinas/química , Lisossomos/metabolismo , Glicoproteínas de Membrana/química , Proteínas de Neoplasias/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
9.
Mol Cell ; 53(4): 562-576, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24508390

RESUMO

The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Regulação Enzimológica da Expressão Gênica , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Proliferação de Células , Cisteína/química , Dissulfetos/química , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Inositol/química , Camundongos , Dados de Sequência Molecular , Oxigênio/química , Fosforilação , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais
10.
J Cell Sci ; 125(Pt 20): 4865-75, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22854046

RESUMO

ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses.


Assuntos
Proteínas de Choque Térmico , Glicoproteínas de Membrana , Dobramento de Proteína , Resposta a Proteínas não Dobradas/genética , Animais , Estresse do Retículo Endoplasmático/genética , Inativação Gênica , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Transdução de Sinais
11.
FASEB J ; 26(9): 3691-702, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22649033

RESUMO

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.


Assuntos
Crescimento , Glicoproteínas de Membrana/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Somatomedinas/antagonistas & inibidores , Animais , Glicemia/análise , Células Cultivadas , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatomedinas/biossíntese
12.
J Am Chem Soc ; 134(23): 9796-804, 2012 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-22642269

RESUMO

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.


Assuntos
Desenho de Drogas , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Drosophila/efeitos dos fármacos , Drosophila/crescimento & desenvolvimento , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Toll-Like/metabolismo
13.
Biochim Biophys Acta ; 1823(3): 774-87, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22079671

RESUMO

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Dobramento de Proteína
14.
Semin Cell Dev Biol ; 21(5): 479-85, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20223290

RESUMO

A system of endoplasmic reticulum (ER) chaperones has evolved to optimize the output of properly folded secretory and membrane proteins. An important player in this network is Glucose Regulated Protein 94 (GRP94). Over the last decade, new structural and functional data have begun to delineate the unique characteristics of GRP94 and have solidified its importance in ER quality control pathways. This review describes our current understanding of GRP94 and the four ways in which it contributes to the ER quality control: (1) chaperoning the folding of proteins; (2) interacting with other components of the ER protein folding machinery; (3) storing calcium; and (4) assisting in the targeting of malfolded proteins to ER-associated degradation (ERAD).


Assuntos
Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Animais , Proteínas de Choque Térmico HSP70 , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
15.
FASEB J ; 24(7): 2292-300, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20181935

RESUMO

MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir-146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir-146a, the chemokine CCL8/MCP-2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication. In the present study, we have analyzed changes in the expression of mir-146a in primary human fetal microglial cells upon infection with HIV-1 and found increased expression of mir-146a. We further show that CCL8/MCP-2 is a target for mir-146a in HIV-1 infected microglia, as overexpression of mir-146a prevented HIV-induced secretion of MCP-2 chemokine. The clinical relevance of our findings was evaluated in HIV-encephalitis (HIVE) brain samples in which decreased levels of MCP-2 and increased levels of mir-146a were observed, suggesting a role for mir-146a in the maintenance of HIV-mediated chronic inflammation of the brain.


Assuntos
Quimiocina CCL8/antagonistas & inibidores , Infecções por HIV/etiologia , HIV-1/patogenicidade , MicroRNAs/genética , Microglia/virologia , Células Cultivadas , Encefalite Viral/patologia , Regulação da Expressão Gênica/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Humanos , Inflamação/virologia
16.
Biochim Biophys Acta ; 1803(2): 333-41, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19914304

RESUMO

The endoplasmic reticulum chaperone GRP94 is essential for early embryonic development and in particular affects differentiation of muscle lineages. To determine why an ubiquitously expressed protein has such a specific effect, we investigated the function of GRP94 in the differentiation of established myogenic cell lines in culture. Using both genetic suppression of expression, via RNA interference, and inhibition of function, via specific chemical inhibitors, we show that GRP94 expression and activity are needed for the in vitro fusion of myoblasts precursors into myotubes and the expression of contractile proteins that mark terminal differentiation. The inhibition can be complemented by addition of insulin-like growth factors to the cultures. GRP94 is not needed for the initial steps of myogenesis, only for the steps downstream of MyoD up-regulation, coinciding with the known need for synergistic input from growth factor signaling. Indeed, GRP94 is needed for the production of insulin-like growth factors I and II (IGF-I and IGF-II) by the differentiating cells. Moreover, the depletion of the chaperone does not increase the rate of apoptosis that always accompanies myogenic differentiation. Thus, the major effect of GRP94 on muscle differentiation is mediated by its regulation of IGF production.


Assuntos
Comunicação Autócrina/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular , Proliferação de Células , Retículo Endoplasmático/metabolismo , Inativação Gênica , Proteínas de Choque Térmico HSP70/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like II/genética , Proteínas de Membrana/genética , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Interferência de RNA
17.
Biochim Biophys Acta ; 1793(2): 378-87, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19022302

RESUMO

HIV-Encephalopathy (HIVE) is a common neurological disorder associated with HIV-1 infection and AIDS. The activity of the HIV trans-activating protein Tat is thought to contribute to neuronal pathogenesis. While Tat proteins from primary virus isolates consist of 101 or more amino acids, 72 and 86 amino acids forms of Tat are commonly used for in vitro studies. Although Tat72 contains the minimal domain required for viral replication, other activities of Tat appear to vary according to its length, sub-cellular localization, cell type and the stage of cellular differentiation. In this study, we investigated the stability of intracellular Tat101 during proliferation and differentiation of neuronal cells in culture. We have utilized rat neuronal progenitors as a model of neuronal cell proliferation and differentiation, as well as rat primary cortical neurons as a model of fully differentiated cells. Our results indicate that, upon internalization, Tat101 was degraded more rapidly in proliferating cells than in cells which either underwent neuronal differentiation or were fully differentiated. Intracellular degradation of Tat was prevented by the calpain 1 inhibitor, ALLN, in both proliferating and differentiated cells. Inhibition of calpain 1 by calpastatin peptide also prevented Tat cleavage. In vitro calpain digestion and mass spectrometry analysis further demonstrated that the sequence of Tat sensitive to calpain cleavage was located in the C-terminus of this viral protein, between amino acids 68 and 69. Moreover, cleavage of Tat101 by calpain 1 increased neurotoxic effect of this viral protein and presence of the calpain inhibitor protected neuronal cells from Tat-mediated toxicity.


Assuntos
Calpaína/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Inibidores Enzimáticos/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Leupeptinas/farmacologia , Dados de Sequência Molecular , Neurônios/citologia , Processamento de Proteína Pós-Traducional , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
18.
J Cell Physiol ; 216(3): 764-70, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18381601

RESUMO

MicroRNAs (miRs) are short endogenous RNAs that regulate gene expression by incomplete pairing with messenger RNAs. An increasing number of studies show that mammalian microRNAs play fundamental roles in various aspects of cellular function including differentiation, proliferation, and cell death. Recent findings demonstrating the presence of microRNAs in mature neuronal dendrites suggest their possible involvement in controlling local protein translation and synaptic function. HIV-1 Encephalopathy (HIVE) is a manifestation of HIV-1 infection that often results in neuronal damage and dysfunction. While neurons are rarely, if ever, infected by HIV-1, they are exposed to cytotoxic viral and cellular factors including the HIV-1 transactivating factor Tat. In this study, we show that Tat deregulates expression levels of selected microRNAs, including the neuronal mir-128, in primary cortical neurons. We further show that mir-128a inhibits expression of the pre-synaptic protein SNAP25, whereas the anti-mir-128a partially restores Tat/mir-128a-induced downregulation of SNAP25 expression. Altogether, our data provide a novel mechanism by which HIV-Tat perturbs neuronal activity.


Assuntos
MicroRNAs/metabolismo , Neurônios/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurônios/citologia , Ratos , Reprodutibilidade dos Testes , Proteína 25 Associada a Sinaptossoma/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
19.
J Cell Physiol ; 215(3): 765-70, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18181169

RESUMO

Docosahexaenoic acid (DHA) is a well known chemopreventive nutrient within diet formulations, but it may also exert toxic effects on cultured cells, while this is limited when also another relevant nutrient as vitamin E is present. This effect, beside the involvement of the two nutrients in oxidative processes, likely affects the expression of specific genes. To obtain information on combined activities of DHA and vitamin E on some gene products previously resulted to be in vivo regulated from dietary unsaturated fats, the effect of the two nutrients was evaluated in human cell line HepG2. Independently, DHA and vitamin E resulted to affect only slightly UDP-glucuronosyltransferase 1A1 (UGT1A1) mRNA expression. Nevertheless, their combination produced a considerable reduction of this mRNA. DHA also downregulated stearoyl-CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP-1) expression, while vitamin E did not affect these products. However, their combination abolished the downregulation of SCD but did not affect that of SREBP-1. Therefore the effect of the two nutrients is related to specific gene regulation processes resulting in a cooperation which might be related to their physiological effects as dietary components.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Vitamina E/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Fluorescência , Glucuronosiltransferase/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
20.
Immunol Lett ; 109(2): 145-54, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17360047

RESUMO

In this study we analysed the regulation of gene expression by arvanil and anandamide in human peripheral blood mononuclear cells (PBMCs) to clarify their immunosuppressive properties. PBMCs were activated, leading to CD36 down regulation, that was normalized by arvanil and anandamide. We used microarray technology to identify a regulatory pattern associated with cell proliferation in the presence of both substances. CD3-CD28 stimulated PBMCs showed a pattern of up-regulated and down-regulated genes after treatment with these substances. We selected and analysed several genes chosen by their function in the regulation of cell proliferation. We showed a transcriptional control of the CD36 gene by arvanil and anandamide associated with an increased protein expression, thus suggesting a possible role of CD36 in anandamide and arvanil anti-inflammatory pattern.


Assuntos
Ácidos Araquidônicos/farmacologia , Antígenos CD36/biossíntese , Capsaicina/análogos & derivados , Leucócitos Mononucleares/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Ácidos Araquidônicos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Antígenos CD36/sangue , Antígenos CD36/imunologia , Capsaicina/imunologia , Capsaicina/farmacologia , Endocanabinoides , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alcamidas Poli-Insaturadas/imunologia , Regulação para Cima/efeitos dos fármacos
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