Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.584
Filtrar
1.
Nat Commun ; 11(1): 3345, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620802

RESUMO

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved RNA decay mechanism that has emerged as a potent cell-intrinsic restriction mechanism of retroviruses and positive-strand RNA viruses. However, whether NMD is capable of restricting DNA viruses is not known. The DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that NMD restricts KSHV lytic reactivation. Leveraging high-throughput transcriptomics we identify NMD targets transcriptome-wide in PEL cells and identify host and viral RNAs as substrates. Moreover, we identified an NMD-regulated link between activation of the unfolded protein response and transcriptional activation of the main KSHV transcription factor RTA, itself an NMD target. Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Viral/metabolismo , Ativação Viral/genética , Linhagem Celular Tumoral , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , RNA Mensageiro/metabolismo , RNA-Seq , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Resposta a Proteínas não Dobradas/genética , Latência Viral/genética
2.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G121-G132, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567324

RESUMO

Nongenomic glucocorticoid (GC) and serum- and glucocorticoid-inducible kinase 1 (SGK1) signaling regulate ion transport, but CFTR has not been investigated in the intestine. We examined GC, SGK1, and phosphatidylinositol 3-kinase (PI3K) kinase signaling of CFTR ion transport in native intestine and the role of GCs on mRNA, protein, surface expression, and cyclic guanosine monophosphate (cGMP)-elicited diarrhea. Rats were treated with dexamethasone (DEXA; 2 mg/kg ip) or DMSO for 1, 4, and 24 h. Cyclic adenosine monophosphate (cAMP)-activated ion transport was examined in the presence or absence of SGK1 and PI3K inhibitors. Phosphorylation of SGK1, phosphoinositide-dependent kinase 1, and Akt kinases was confirmed by immunoblots using phosphor-specific antibodies. Tissue lysates were analyzed by mass spectrometry. CFTR and SGK1 mRNA were measured by quantitative PCR. Changes in total and surface CFTR protein were determined. The role of GC in cGMP-activated CFTR ion transport was examined. GC synergistically increased CFTR ion transport by SGK1 and PI3K signaling and increased CFTR protein without altering SGK1 or CFTR mRNA. GC induced highest levels of CFTR protein at 4 h that were associated with marked increase in surface CFTR, phosphorylation of the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-like (Nedd4-2), and 14-3-3ε, supporting their roles in surface retention and stability. Coimmunoprecipitation of CFTR, Nedd4-2, and 14-3-3ε indicated that assembly of this complex is a likely effector of the SGK and Akt pathways. Mass spectrometry identified phosphorylated peptides in relevant proteins. GC-SGK1 potently regulates CFTR in the intestine and is implicated in diarrheal disease.NEW & NOTEWORTHY This is the first study to examine the mechanisms of glucocorticoid, serum- and glucocorticoid-inducible kinase 1, and nongenomic kinase signaling of CFTR in the native intestine. We identified unique and druggable intestine-specific factors of the pathway that are targets for treating stress-induced diarrhea.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dexametasona/toxicidade , Diarreia/etiologia , Dimetil Sulfóxido/toxicidade , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Diarreia/induzido quimicamente , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Masculino , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinase Piruvato Desidrogenase (Transferência de Acetil)/genética , Quinase Piruvato Desidrogenase (Transferência de Acetil)/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismo
3.
Anticancer Res ; 40(6): 3255-3264, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487620

RESUMO

BACKGROUND/AIM: Rta, a transactivator of Epstein-Barr virus, is associated with progression of nasopharyngel carcinoma (NPC); however, its mechanism of contribution to the pathogenesis of NPC remains unclear. Interleukin-6 (IL-6), a tumor promoter, is detected in NPC. This in vitro study examined whether and how Rta promotes NPC progression by up-regulating IL-6. MATERIALS AND METHODS: Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, ELISA, immunoblotting assays, reporter gene assays, and transwell migration assays were performed. RESULTS: In NPC cells, Rta up-regulated IL-6 expression at the mRNA and protein levels, and the Rta's C-terminus was essential for promoter activation and expression of IL-6. The induction of IL-6 by Rta also required activation of extracellular signal-regulated kinase 1/2 and activator protein-1. Furthermore, IL-6 secreted from Rta-expressing NPC cells promoted migration of Rta-negative NPC cells by activating IL-6 receptor/Janus kinase/signal transducer and activator of transcription 3 pathway. CONCLUSION: Rta contributes to progression of NPC cells through induction of IL-6 in vitro.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Neoplasias/metabolismo , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transativadores/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/virologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologia , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia , Transativadores/genética , Fator de Transcrição AP-1/metabolismo , Transfecção , Regulação para Cima
4.
PLoS Pathog ; 16(5): e1008537, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365141

RESUMO

Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.


Assuntos
Infecções por Citomegalovirus , Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces , Imunidade Inata , Proteína da Leucemia Promielocítica , Replicação Viral , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Regulação Viral da Expressão Gênica/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/imunologia , Mutação , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/imunologia , Sumoilação/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
5.
Metabolism ; 107: 154222, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32246987

RESUMO

Fructose over-consumption contributes to the development of liver steatosis in part by stimulating ChREBPα-driven de novo lipogenesis. However, the mechanisms by which fructose activates ChREBP pathway remain largely undefined. Here we performed affinity purification of ChREBPα followed by mass spectrometry and identified DDB1 as a novel interaction protein of ChREBPα in the presence of fructose. Depletion and overexpression of Ddb1 showed opposite effects on the ChREBPα stability in hepatocytes. We next tested the impact of hepatic Ddb1 deficiency on the fructose-induced ChREBP pathway. After 3-week high-fructose diet feeding, both Ddb1 liver-specific knockout and AAV-TBG-Cre-injected Ddb1flox/flox mice showed significantly reduced ChREBPα, lipogenic enzymes, as well as triglycerides in the liver. Mechanistically, DDB1 stabilizes ChREBPα through CRY1, a known ubiquitination target of DDB1 E3 ligase. Finally, overexpression of a degradation-resistant CRY1 mutant (CRY1-585KA) reduces ChREBPα and its target genes in the mouse liver following high-fructose diet feeding. Our data revealed DDB1 as an intracellular sensor of fructose intake to promote hepatic de novo lipogenesis and liver steatosis by stabilizing ChREBPα in a CRY1-dependent manner.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Criptocromos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Carboidratos da Dieta/farmacologia , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Frutose/farmacologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana/metabolismo , Animais , Hepatócitos/metabolismo , Proteínas Imediatamente Precoces/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Cultura Primária de Células , Ubiquitinação
6.
PLoS Pathog ; 16(4): e1008496, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32320442

RESUMO

Human herpesviruses 6A and 6B (HHV-6A/B) are unique among human herpesviruses in their ability to integrate their genome into host chromosomes. Viral integration occurs at the ends of chromosomes within the host telomeres. The ends of the HHV-6A/B genomes contain telomeric repeats that facilitate the integration process. Here, we report that productive infections are associated with a massive increase in telomeric sequences of viral origin. The majority of the viral telomeric signals can be detected within viral replication compartments (VRC) that contain the viral DNA processivity factor p41 and the viral immediate-early 2 (IE2) protein. Components of the shelterin protein complex present at telomeres, including TRF1 and TRF2 are also recruited to VRC during infection. Biochemical, immunofluorescence coupled with in situ hybridization and chromatin immunoprecipitation demonstrated the binding of TRF2 to the HHV-6A/B telomeric repeats. In addition, approximately 60% of the viral IE2 protein localize at cellular telomeres during infection. Transient knockdown of TRF2 resulted in greatly reduced (13%) localization of IE2 at cellular telomeres (p<0.0001). Lastly, TRF2 knockdown reduced HHV-6A/B integration frequency (p<0.05), while no effect was observed on the infection efficiency. Overall, our study identified that HHV-6A/B IE2 localizes to telomeres during infection and highlight the role of TRF2 in HHV-6A/B infection and chromosomal integration.


Assuntos
Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Integração Viral/genética , Linhagem Celular Tumoral , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Infecções por Roseolovirus/genética , Infecções por Roseolovirus/metabolismo , Infecções por Roseolovirus/virologia , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
7.
PLoS Pathog ; 16(4): e1008390, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294138

RESUMO

Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication (oriLyt) and forms an RNA-DNA hybrid (R-loop) through its G+C-rich 5' end, which may be important for the initiation of viral DNA replication. Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among betaherpesviruses.


Assuntos
Citomegalovirus/genética , Replicação do DNA , DNA Viral/genética , Proteínas Imediatamente Precoces/metabolismo , RNA Longo não Codificante/genética , Proteínas Virais/genética , Replicação Viral , Animais , Células Cultivadas , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/microbiologia , Infecções por Citomegalovirus/patologia , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Origem de Replicação
8.
PLoS Pathog ; 16(4): e1008426, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282833

RESUMO

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/ß/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection.


Assuntos
Infecções por Citomegalovirus/enzimologia , Citomegalovirus/metabolismo , Granzimas/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Células Matadoras Naturais/enzimologia , Transativadores/metabolismo , Motivos de Aminoácidos , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Granzimas/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/genética , Proteólise , Linfócitos T Citotóxicos/enzimologia , Transativadores/genética
9.
PLoS Pathog ; 16(4): e1008402, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251483

RESUMO

Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, late in infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection using dTag methodology and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that in late infection the IE2 proteins target a distinct subset of HCMV early-late and late promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/metabolismo , Replicação do DNA , Proteínas Imediatamente Precoces/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transativadores/metabolismo , Proteínas Virais/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , DNA Viral/genética , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , RNA Polimerase II/genética , Transativadores/genética , Iniciação da Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral
10.
Science ; 367(6483): 1255-1260, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165587

RESUMO

T cells maintain a quiescent state prior to activation. As inappropriate T cell activation can cause disease, T cell quiescence must be preserved. Despite its importance, the mechanisms underlying the "quiescent state" remain elusive. Here, we identify BTG1 and BTG2 (BTG1/2) as factors responsible for T cell quiescence. BTG1/2-deficient T cells show an increased proliferation and spontaneous activation due to a global increase in messenger RNA (mRNA) abundance, which reduces the threshold to activation. BTG1/2 deficiency leads to an increase in polyadenylate tail length, resulting in a greater mRNA half-life. Thus, BTG1/2 promote the deadenylation and degradation of mRNA to secure T cell quiescence. Our study reveals a key mechanism underlying T cell quiescence and suggests that low mRNA abundance is a crucial feature for maintaining quiescence.


Assuntos
Proteínas Imediatamente Precoces/fisiologia , Ativação Linfocitária , Proteínas de Neoplasias/fisiologia , Estabilidade de RNA , RNA Mensageiro/química , Linfócitos T/imunologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Células Cultivadas , Proteínas Imediatamente Precoces/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Poliadenilação , Proteínas Supressoras de Tumor/genética
11.
Neoplasma ; 67(3): 614-622, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32009420

RESUMO

Human immediate early response 2 (IER2) has been implicated in tumor cell motility and metastasis; however, the underlying mechanisms in hepatocellular carcinoma (HCC) metastasis remain to be clarified. In this study, we demonstrate that dysregulation of IER2 was shown in HCC clinical samples, and IER2 expression resulted in the promotion of cell migration and invasion in vitro, and HCC tumor growth and pulmonary metastasis in vivo. Moreover, we showed that IER2 expression altered assembly of the actin cytoskeleton rearrangement. Furthermore, MAPK and PI3K/Akt signaling pathways induced by IER2 were confirmed to be probably involved in regulating the activity of Rho GTPases, such as RhoA, Rac1 and Cdc42. Collectively, our results indicated a significant role of IER2 in the HCC cell motility and metastasis through MAPK and PI3K/Akt signaling pathways to regulate the activity of Rho GTPases, thereby modulating actin cytoskeleton rearrangement, unveiling a novel mechanism of cell motility regulation induced by IER2.


Assuntos
Carcinoma Hepatocelular/patologia , Proteínas Imediatamente Precoces/genética , Neoplasias Hepáticas/patologia , Transativadores/genética , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias Hepáticas/genética , Fosfatidilinositol 3-Quinases , Transdução de Sinais
12.
Biochem Biophys Res Commun ; 524(4): 910-915, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32051088

RESUMO

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Domínio Catalítico , Cromonas/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Degradação Associada com o Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Leupeptinas/farmacologia , Morfolinas/farmacologia , Estresse Nitrosativo , Compostos Nitrosos/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/genética , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32102879

RESUMO

Lipoxin A4 (LXA4) is an endogenous lipid mediator with compelling anti-inflammatory and proresolution properties. Studies done to assess the role of arachidonic acid pathways of the host in Kaposi's sarcoma-associated herpesvirus (KSHV) biology helped discover that KSHV infection hijacks the proinflammatory cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) pathways and concurrently reduces anti-inflammatory LXA4 secretion to maintain KSHV latency in infected cells. Treatment of KSHV-infected cells with LXA4 minimizes the activation of inflammatory and proliferative signaling pathways, including the NF-κB, AKT, and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, but the exact mechanism of action of LXA4 remains unexplored. Here, using mass spectrometry analysis, we identified components from the minichromosome maintenance (MCM) protein and chromatin-remodeling complex SMARCB1 and SMARCC2 to be LXA4-interacting host proteins in KSHV-infected cells. We identified a higher level of nuclear aryl hydrocarbon receptor (AhR) in LXA4-treated KSHV-infected cells than in untreated KSHV-infected cells, which probably facilitates the affinity interaction of the nucleosome complex protein with LXA4. We demonstrate that SMARCB1 regulates both replication and transcription activator (RTA) activity and host hedgehog (hh) signaling in LXA4-treated KSHV-infected cells. Host hedgehog signaling was modulated in an AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-S6 kinase-dependent manner in LXA4-treated KSHV-infected cells. Since anti-inflammatory drugs are beneficial as adjuvants to conventional and immune-based therapies, we evaluated the potential of LXA4 treatment in regulating programmed death-ligand 1 (PD-L1) on KSHV-carrying tumor cells. Overall, our study identified LXA4-interacting host factors in KSHV-infected cells, which could help provide an understanding of the mode of action of LXA4 and its therapeutic potential against KSHV.IMPORTANCE The latent-to-lytic switch in KSHV infection is one of the critical events regulated by the major replication and transcription activator KSHV protein called RTA. Chromatin modification of the viral genome determines the phase of the viral life cycle in the host. Here, we report that LXA4 interacts with a host chromatin modulator, especially SMARCB1, which upregulates the KSHV ORF50 promoter. SMARCB1 has also been recognized to be a tumor suppressor protein which controls many tumorigenic events associated with the hedgehog (hh) signaling pathway. We also observed that LXA4 treatment reduces PD-L1 expression and that PD-L1 expression is an important immune evasion strategy used by KSHV for its survival and maintenance in the host. Our study underscores the role of LXA4 in KSHV biology and emphasizes that KSHV is strategic in downregulating LXA4 secretion in the host to establish latency. This study also uncovers the therapeutic potential of LXA4 and its targetable receptor, AhR, in KSHV's pathogenesis.


Assuntos
Cromatina/fisiologia , Herpesvirus Humano 8/metabolismo , Lipoxinas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Araquidonato 5-Lipoxigenase/genética , Antígeno B7-H1/metabolismo , Linhagem Celular , Cromatina/metabolismo , Ciclo-Oxigenase 2/metabolismo , Regulação Viral da Expressão Gênica/genética , Proteínas Hedgehog/metabolismo , Herpesvirus Humano 8/patogenicidade , Proteínas Imediatamente Precoces/genética , Lipoxinas/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína SMARCB1/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Transativadores/metabolismo , Latência Viral/genética , Replicação Viral/genética
14.
Cell ; 180(5): 878-894.e19, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32059783

RESUMO

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.


Assuntos
Autoanticorpos/genética , Doenças Autoimunes/genética , Linfócitos B/imunologia , Linfoma/genética , Animais , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Transporte/genética , Evolução Clonal/genética , Evolução Clonal/imunologia , Ciclina D3/genética , Guanilato Ciclase/genética , Humanos , Proteínas Imediatamente Precoces/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Proteínas Inibidoras de Diferenciação/genética , Linfoma/imunologia , Linfoma/patologia , Camundongos , Mutação/genética , Mutação/imunologia , Proteínas de Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteínas Supressoras de Tumor/genética , Recombinação V(D)J/genética
15.
Nat Commun ; 11(1): 293, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941886

RESUMO

Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3' processing factor CPSF. It thereby induces the assembly of a dead-end 3' processing complex, blocking mRNA 3' cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3' processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer.


Assuntos
Herpesvirus Humano 1/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Terminação da Transcrição Genética , Animais , Linhagem Celular , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Células HeLa , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética
16.
PLoS Pathog ; 16(1): e1008268, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923286

RESUMO

Establishment of viral latency is not only essential for lifelong Kaposi's sarcoma-associated herpesvirus (KSHV) infection, but it is also a prerequisite of viral tumorigenesis. The latent viral DNA has a complex chromatin structure, which is established in a stepwise manner regulated by host epigenetic factors during de novo infection. However, despite the importance of viral latency in KSHV pathogenesis, we still have limited information about the repertoire of epigenetic factors that are critical for the establishment and maintenance of KSHV latency. Therefore, the goal of this study was to identify host epigenetic factors that suppress lytic KSHV genes during primary viral infection, which would indicate their role in latency establishment. We performed an siRNA screen targeting 392 host epigenetic factors during primary infection and analyzed which ones affect the expression of the viral replication and transcription activator (RTA) and/or the latency-associated nuclear antigen (LANA), which are viral genes essential for lytic replication and latency, respectively. As a result, we identified the Nucleosome Remodeling and Deacetylase (NuRD) complex, Tip60 and Tip60-associated co-repressors, and the histone demethylase KDM2B as repressors of KSHV lytic genes during both de novo infection and the maintenance of viral latency. Furthermore, we showed that KDM2B rapidly binds to the incoming viral DNA as early as 8 hpi, and can limit the enrichment of activating histone marks on the RTA promoter favoring the downregulation of RTA expression even prior to the polycomb proteins-regulated heterochromatin establishment on the viral genome. Strikingly, KDM2B can also suppress viral gene expression and replication during lytic infection of primary gingival epithelial cells, revealing that KDM2B can act as a host restriction factor of the lytic cycle of KSHV during both latent and lytic infections in multiple different cell types.


Assuntos
Infecções por Herpesviridae/genética , Herpesvirus Humano 8/fisiologia , RNA Interferente Pequeno/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Epigênese Genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina Acetiltransferase 5/genética , Lisina Acetiltransferase 5/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Transativadores/metabolismo , Latência Viral
17.
J Gen Virol ; 101(4): 420-425, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31985394

RESUMO

The γ-herpesviruses have proved hard to vaccination against, with no convincing protection against long-term latent infection by recombinant viral subunits. In experimental settings, whole-virus vaccines have proved more effective, even when the vaccine virus itself establishes latent infection poorly. The main alternative is replication-deficient virus particles. Here high-dose, replication-deficient murid herpesvirus-4 only protected mice partially against wild-type infection. By contrast, latency-deficient but replication-competent vaccine protected mice strongly, even when delivered non-invasively to the olfactory epithelium. Thus, this approach seems to provide the best chance of a safe and effective γ-herpesvirus vaccine.


Assuntos
Infecções por Herpesviridae/prevenção & controle , Rhadinovirus/imunologia , Vacinas Virais , Animais , Anticorpos Antivirais/sangue , Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/imunologia , Proteínas Imediatamente Precoces/genética , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Transativadores/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vírion/imunologia , Latência Viral/imunologia , Replicação Viral/genética
18.
J Pharm Pharmacol ; 72(1): 68-75, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31721211

RESUMO

OBJECTIVES: MicroRNAs are abundant in eukaryotic cells and play key roles in cancers. Circular RNAs (CircRNAs) served as the competing endogenous RNAs (ceRNAs) in mediating multiple cell processes. This study aims to define the role of CircRNA CircZNF609/miR-134-5p in glioma as well as the underlying regulating mechanism. METHODS: Relative expression of miR-134-5p, CircZNF609 and BTG-2 mRNA was determined by quantitative real-time PCR. Cell proliferation was analysed by CCK-8 assay. Cell migration was assessed by cell wound scratch assay. The direct regulatory of miR-134-5p on BTG-2 and CircZNF609 was verified by luciferase report gene assay. KEY FINDINGS: MiR-134-5p was significantly upregulated in glioma cells. The overexpression of miR-134-5p inhibited cell proliferation and migration of glioma cell U251 and U87. Reversely, knock-down of miR-134-5p enhanced cell proliferation and migration. Both BTG-2 and CircZNF609 are the direct targets of miR-134-5p, and their expression could be negatively regulated by miR-134-5p. CircZNF609 was significantly upregulated in U251 and U87 cells and acted as an oncogene to promote cell proliferation and cell migration of glioma cell U251 and U87. CONCLUSIONS: These data proved that CircZNF609 served as a competing RNA to bind miR-134-5p that promoted BTG-2 expression leading to reduced proliferation and migration of glioma cell.


Assuntos
Neoplasias Encefálicas/metabolismo , Movimento Celular , Proliferação de Células , Glioma/metabolismo , Proteínas Imediatamente Precoces/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Humanos , Proteínas Imediatamente Precoces/genética , MicroRNAs/genética , RNA Circular/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética
19.
Biosci Trends ; 13(6): 502-509, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31866613

RESUMO

Although cytomegalovirus (HCMV) infection is asymptomatic in healthy individuals, the virus can remain latent for many years due to its ability to evade host immune surveillance. However, reactivation of HCMV can lead to life-threatening disease. Recent studies have shown that HCMV infection mediates immune escape by regulating macrophage activity, although the role of the HCMV-encoded IE2 protein is unclear. A ul122 transgenic mouse model was created to stably expresses the IE2 protein, and the proportion of M1 and M2 macrophage populations in their spleen and bone marrow was compared to that in wild-type controls. In addition, the phagocytic function of the macrophages was evaluated in terms of neutral red dye uptake. Spleen and bone marrow macrophages in IE2-expressing mice were mainly of the M2 phenotype and displayed enhanced phagocytic function compared to that in control mice. The relative levels of expression of macrophage-related GRB2 and of IL-4, IFN-γ, IL-13, and TNF-α were also analyzed in the spleen and bone marrow of the two groups. The IE2-expressing mice had increased expression of GRB2 and increased expression of the M2-related cytokines IL-4 and IL-13. Taken together, the current results suggest that HCMV IE2 polarizes the host macrophages to the M2 type via a GRB2/IL-4-related pathway, which enables long-term survival of the virus in the host.


Assuntos
Proteína Adaptadora GRB2/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Transativadores/metabolismo , Animais , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Proteína Adaptadora GRB2/genética , Proteínas Imediatamente Precoces/genética , Macrófagos/citologia , Masculino , Camundongos , Camundongos Transgênicos , Fagocitose/genética , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/genética
20.
PLoS Pathog ; 15(11): e1008122, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765434

RESUMO

The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαß sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRß chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRß, in understanding the CD8 T cell receptor repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Proteínas Imediatamente Precoces/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Transativadores/imunologia , Sequência de Aminoácidos , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígeno HLA-A2/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transativadores/genética , Transativadores/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA