RESUMO
Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.
Assuntos
Proliferação de Células/genética , Ciclina D3/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Proteínas Imediatamente Precoces/genética , Linfopoese/genética , Células Precursoras de Linfócitos B/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas Supressoras de Tumor/genética , Animais , Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Rearranjo Gênico do Linfócito B/genética , Genes abl/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Cadeias Leves de Imunoglobulina/genética , Espectrometria de Massas , Camundongos , Células Precursoras de Linfócitos B/citologia , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Supressoras de Tumor/metabolismoRESUMO
Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células , Proteínas Imediatamente Precoces/metabolismo , Mitose , Células Neoplásicas Circulantes/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Regiões 3' não Traduzidas , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Montagem e Desmontagem da Cromatina , Feminino , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Indóis/farmacologia , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Mitose/efeitos dos fármacos , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fenilacetatos/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estruturas R-Loop , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais , Elongação da Transcrição Genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient â¼7) generated by homomultimerization of the virus's essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Redes Reguladoras de Genes , Aptidão Genética , Citomegalovirus/fisiologia , Retroalimentação Fisiológica , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Corpos de Inclusão Viral/metabolismo , Imagem com Lapso de Tempo , Transativadores/metabolismo , Ativação Transcricional , Replicação ViralRESUMO
Deubiquitinases (DUBs) remove ubiquitin from substrates and play crucial roles in diverse biological processes. However, our understanding of deubiquitination in viral replication remains limited. Employing an oncogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) to probe the role of protein deubiquitination, we found that Ovarian tumor family deubiquitinase 4 (OTUD4) promotes KSHV reactivation. OTUD4 interacts with the replication and transcription activator (K-RTA), a key transcription factor that controls KSHV reactivation, and enhances K-RTA stability by promoting its deubiquitination. Notably, the DUB activity of OTUD4 is not required for K-RTA stabilization; instead, OTUD4 functions as an adaptor protein to recruit another DUB, USP7, to deubiquitinate K-RTA and facilitate KSHV lytic reactivation. Our study has revealed a novel mechanism whereby KSHV hijacks OTUD4-USP7 deubiquitinases to promote lytic reactivation, which could be potentially harnessed for the development of new antiviral therapies.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Sarcoma de Kaposi , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Transativadores/genética , Herpesvirus Humano 8/genética , Replicação Viral , Regulação Viral da Expressão Gênica , Ativação Viral , Proteases Específicas de Ubiquitina/metabolismoRESUMO
SGK1 is an AGC kinase that regulates the expression of membrane sodium channels in renal tubular cells in a manner dependent on the metabolic checkpoint kinase complex mTORC2. We hypothesized that SGK1 might represent an additional mTORC2-dependent regulator of the differentiation and function of T cells. Here we found that after activation by mTORC2, SGK1 promoted T helper type 2 (TH2) differentiation by negatively regulating degradation of the transcription factor JunB mediated by the E3 ligase Nedd4-2. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling expression of the long isoform of the transcription factor TCF-1. Consistent with those findings, mice with selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated substantial IFN-γ in response to viral infection and more readily rejected tumors.
Assuntos
Asma/imunologia , Proteínas Imediatamente Precoces/metabolismo , Melanoma Experimental/imunologia , Complexos Multiproteicos/imunologia , Infecções por Poxviridae/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR/imunologia , Células Th1/imunologia , Células Th2/imunologia , Vaccinia virus/imunologia , Imunidade Adaptativa/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica/genética , Fator 1-alfa Nuclear de Hepatócito , Proteínas Imediatamente Precoces/genética , Interferon gama/genética , Interferon gama/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4 , Proteínas Serina-Treonina Quinases/genética , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Tumoral/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.
Assuntos
Herpesvirus Humano 6 , Proteínas Imediatamente Precoces , Infecções por Roseolovirus , Humanos , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , Infecções por Roseolovirus/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Integração Viral , Instabilidade Genômica , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Interferons (IFNs) and the products of interferon-stimulated genes (ISGs) play crucial roles in host defense against virus infections. Although many ISGs have been characterized with respect to their antiviral activity, their target specificities and mechanisms of action remain largely unknown. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is linked to several human malignancies. Here, we used the genetically and biologically related virus, murine gammaherpesvirus 68 (MHV-68) and screened for ISGs with anti-gammaherpesvirus activities. We found that overexpression of RNF213 dramatically inhibited MHV-68 infection, whereas knockdown of endogenous RNF213 significantly promoted MHV-68 proliferation. Importantly, RNF213 also inhibited KSHV de novo infection, and depletion of RNF213 in the latently KSHV-infected iSLK-219 cell line significantly enhanced lytic reactivation. Mechanistically, we demonstrated that RNF213 targeted the Replication and Transcription Activator (RTA) of both KSHV and MHV-68, and promoted the degradation of RTA protein through the proteasome-dependent pathway. RNF213 directly interacted with RTA and functioned as an E3 ligase to ubiquitinate RTA via K48 linkage. Taken together, we conclude that RNF213 serves as an E3 ligase and inhibits the de novo infection and lytic reactivation of gammaherpesviruses by degrading RTA through the ubiquitin-proteasome pathway.
Assuntos
Gammaherpesvirinae , Infecções por Herpesviridae , Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Humanos , Adenosina Trifosfatases/metabolismo , Gammaherpesvirinae/genética , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Herpesvirus Humano 8/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Latência Viral/genética , Replicação ViralRESUMO
The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Histonas/genética , Histonas/metabolismo , Nucleossomos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Latência Viral/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Sequências Repetidas Terminais/genética , Regulação Viral da Expressão GênicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.
Assuntos
Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8 , Regiões Promotoras Genéticas , Ativação Viral , Latência Viral , Humanos , Antígenos Virais/genética , Antígenos Virais/metabolismo , Cromatina/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sequências Repetidas Terminais/genética , Transativadores/metabolismo , Ativação Viral/genética , Latência Viral/genéticaRESUMO
Placental infection plays a central role in the pathogenesis of congenital human cytomegalovirus (HCMV) infections and is a cause of fetal growth restriction and pregnancy loss. HCMV can replicate in some trophoblast cell types, but it remains unclear how the virus evades antiviral immunity in the placenta and how infection compromises placental development and function. Human trophoblast stem cells (TSCs) can be differentiated into extravillous trophoblasts (EVTs), syncytiotrophoblasts (STBs), and organoids, and this study assessed the utility of TSCs as a model of HCMV infection in the first-trimester placenta. HCMV was found to non-productively infect TSCs, EVTs, and STBs. Immunofluorescence assays and flow cytometry experiments further revealed that infected TSCs frequently only express immediate early viral gene products. Similarly, RNA sequencing found that viral gene expression in TSCs does not follow the kinetic patterns observed during lytic infection in fibroblasts. Canonical antiviral responses were largely not observed in HCMV-infected TSCs and TSC-derived trophoblasts. Rather, infection dysregulated factors involved in cell identity, differentiation, and Wingless/Integrated signaling. Thus, while HCMV does not replicate in TSCs, infection may perturb trophoblast differentiation in ways that could interfere with placental function. IMPORTANCE: Placental infection plays a central role in human cytomegalovirus (HCMV) pathogenesis during pregnancy, but the species specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human trophoblast stem cells (TSCs) represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.
Assuntos
Citomegalovirus , Células-Tronco , Trofoblastos , Replicação Viral , Feminino , Humanos , Gravidez , Diferenciação Celular/genética , Linhagem da Célula/genética , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/fisiopatologia , Infecções por Citomegalovirus/virologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Placenta/citologia , Placenta/patologia , Placenta/fisiopatologia , Placenta/virologia , Primeiro Trimestre da Gravidez , Células-Tronco/citologia , Células-Tronco/virologia , Trofoblastos/citologia , Trofoblastos/virologiaRESUMO
TRIM32 is often aberrantly expressed in many types of cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi's sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE: TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi's sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Proteólise , Transativadores , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Ativação Viral , Replicação Viral , Humanos , Apoptose , Linhagem Celular , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Linfoma de Efusão Primária/virologia , Linfoma de Efusão Primária/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/metabolismo , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Latência ViralRESUMO
The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment.
Assuntos
Glioma , Proteínas Imediatamente Precoces , Animais , Humanos , Camundongos , Citomegalovirus/fisiologia , Regulação para Baixo , Expressão Gênica , Glioma/genética , Glioma/patologia , Proteínas Imediatamente Precoces/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.
Assuntos
Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Multimerização Proteica , Ubiquitina-Proteína Ligases , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Cristalografia por Raios X , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Modelos Moleculares , Humanos , Domínios Proteicos , Dobramento de Proteína , Sequência de Aminoácidos , Conformação Proteica em Folha betaRESUMO
OBJECTIVES: Serum/glucocorticoid-inducible kinase 1 (SGK1) gene encodes a serine/threonine protein kinase that plays an essential role in cellular stress response and regulation of multiple metabolic processes. However, its role in bovine adipogenesis remains unknown. In this study, we aimed to clarify the role of SGK1 in bovine lipid accumulation and improvement of meat quality. METHODS: Preadipocytes were induced to differentiation to detect the temporal expression pattern of SGK1. Heart, liver, lung, spleen, kidney, muscle and fat tissues were collected to detect its tissue expression profile. Recombinant adenovirus and the lentivirus were packaged for overexpression and knockdown. Oil Red O staining, quantitative real-time PCR, Western blot analysis, Yeast two-hybrid assay, luciferase assay and RNA-seq were performed to study the regulatory mechanism of SGK1. RESULTS: SGK1 showed significantly higher expression in adipose and significantly induced expression in differentiated adipocytes. Furthermore, overexpression of SGK1 greatly promoted adipogenesis and inhibited proliferation, which could be shown by the remarkable increasement of lipid droplet, and the expression levels of adipogenic marker genes and cell cycle-related genes. Inversely, its knockdown inhibited adipogenesis and facilitated proliferation. Mechanistically, SGK1 regulates the phosphorylation and expression of two critical proteins of FoxO family, FOXO1/FOXO3. Importantly, SGK1 attenuates the transcriptional repression role of FOXO1 for PPARγ via phosphorylating the site S256, then promoting the bovine fat deposition. CONCLUSIONS: SGK1 is a required epigenetic regulatory factor for bovine preadipocyte proliferation and differentiation, which contributes to a better understanding of fat deposition and meat quality improvement in cattle.
Assuntos
Adipócitos , Adipogenia , Proteína Forkhead Box O1 , Proteínas Imediatamente Precoces , Metabolismo dos Lipídeos , Proteínas Serina-Treonina Quinases , Animais , Bovinos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Adipócitos/metabolismo , Adipócitos/citologia , Adipogenia/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Diferenciação Celular , Proliferação de Células , Regulação da Expressão GênicaRESUMO
Mammalian (or mechanistic) target of rapamycin complex 2 (mTORC2) is a kinase complex that targets predominantly Akt family proteins, SGK1 and protein kinase C (PKC), and has well-characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular K+ concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl-), which stimulates a previously unknown scaffolding activity of the protein 'with no lysine-1' (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2, resulting in enhanced SGK1 phosphorylation and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1-dependent regulatory mechanism that harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.
Assuntos
Proteínas Imediatamente Precoces , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Cloretos/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Proteínas Serina-Treonina Quinases , Serina-Treonina Quinases TOR/metabolismo , Transporte de Íons , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mamíferos/metabolismoRESUMO
Abundant evidence has shown the protective effect of aerobic exercise on central neuronal system, however, research about resistance exercise remains limited. To evaluate the effect and potential molecular mechanisms of resistance exercise in improving cognition and mental health, three-month-old male C57BL/6J mice underwent resistance training for five weeks. Body parameters, cognitive performance and synaptic plasticity were then assessed. In both groups, total RNA from the frontal cortex, hippocampus and gastrocnemius was isolated and sequenced, GO term and KEGG analysis were performed to identify molecular mechanisms. The results from RNA sequencing were then verified by RT-PCR. Our data found that mice in training group showed reduced anxiety-like behavior and better spatial memory. Accordingly, resistance exercise specifically increased the number of thin spines without affecting the number of other kind of spines. mRNA sequence analysis showed that resistance exercise induced differential expression of hundreds of genes in the above three tissues. KEGG analysis indicated the FoxO signaling pathway the most significant changed pathway throughout the brain and muscle. GO terms analysis showed that Sgk1 was enriched in the three key cognition related BP, including long-term memory, learning or memory and memory, and the expression level of Sgk1 was positive related with cognitive performance in the water maze. In conclusion, resistance exercise improved the mental health, cognition and synaptic plasticity of mice. Integrating analysis of mRNA expression profiles in frontal cortex, hippocampus and muscle reveals Sgk1 as the key mediator in brain-muscle crosstalk.
Assuntos
Encéfalo , Proteínas Imediatamente Precoces , Camundongos Endogâmicos C57BL , Músculo Esquelético , Condicionamento Físico Animal , Proteínas Serina-Treonina Quinases , RNA Mensageiro , Animais , Masculino , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Encéfalo/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Treinamento Resistido , Cognição/fisiologia , Transcriptoma , Plasticidade Neuronal/genética , Hipocampo/metabolismo , Ansiedade/genética , Ansiedade/metabolismoRESUMO
IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Transcrição Gênica , Humanos , Genes Virais/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/metabolismo , Iniciação da Transcrição Genética , Elongação da Transcrição Genética , Terminação da Transcrição GenéticaRESUMO
Viperin is a multifunctional interferon-inducible protein that is directly induced in cells by human cytomegalovirus (HCMV) infection. The viral mitochondrion-localized inhibitor of apoptosis (vMIA) interacts with viperin at the early stages of infection and translocates it from the endoplasmic reticulum to the mitochondria, where viperin modulates the cellular metabolism to increase viral infectivity. Viperin finally relocalizes to the viral assembly compartment (AC) at late stages of infection. Despite the importance of vMIA interactions with viperin during viral infection, their interacting residues are unknown. In the present study, we showed that cysteine residue 44 (Cys44) of vMIA and the N-terminal domain (amino acids [aa] 1 to 42) of viperin are necessary for their interaction and for the mitochondrial localization of viperin. In addition, the N-terminal domain of mouse viperin, which is structurally similar to that of human viperin, interacted with vMIA. This indicates that the structure, rather than the sequence composition, of the N-terminal domain of viperin, is required for the interaction with vMIA. Recombinant HCMV, in which Cys44 of vMIA was replaced by an alanine residue, failed to translocate viperin to the mitochondria at the early stages of infection and inefficiently relocalized it to the AC at late stages of infection, resulting in the impairment of viperin-mediated lipid synthesis and a reduction in viral replication. These data indicate that Cys44 of vMIA is therefore essential for the intracellular trafficking and function of viperin to increase viral replication. Our findings also suggest that the interacting residues of these two proteins are potential therapeutic targets for HCMV-associated diseases. IMPORTANCE Viperin traffics to the endoplasmic reticulum (ER), mitochondria, and viral assembly compartment (AC) during human cytomegalovirus (HCMV) infection. Viperin has antiviral activity at the ER and regulates cellular metabolism at the mitochondria. Here, we show that Cys44 of HCMV vMIA protein and the N-terminal domain (aa 1 to 42) of viperin are necessary for their interaction. Cys44 of vMIA also has a critical role for viperin trafficking from the ER to the AC via the mitochondria during viral infection. Recombinant HCMV expressing a mutant vMIA Cys44 has impaired lipid synthesis and viral infectivity, which are attributed to mislocalization of viperin. Cys44 of vMIA is essential for the trafficking and function of viperin and may be a therapeutic target for HCMV-associated diseases.
Assuntos
Proteínas Imediatamente Precoces , Proteína Viperina , Proteínas Virais , Viroses , Animais , Humanos , Camundongos , Cisteína/metabolismo , Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Lipídeos , Mitocôndrias/metabolismo , Viroses/metabolismo , Proteína Viperina/metabolismo , Proteínas Virais/metabolismoRESUMO
IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing human herpesvirus that establishes a persistent infection in humans. The lytic viral cycle plays a crucial part in lifelong infection as it is involved in the viral dissemination. The master regulator of the KSHV lytic replication cycle is the viral replication and transcription activator (RTA) protein, which is necessary and sufficient to push the virus from latency into the lytic phase. Thus, the identification of host factors utilized by RTA for controlling the lytic cycle can help to find novel targets that could be used for the development of antiviral therapies against KSHV. Using a proteomics approach, we have identified a novel interaction between RTA and the cellular E3 ubiquitin ligase complex RNF20/40, which we have shown to be necessary for promoting RTA-induced KSHV lytic cycle.
Assuntos
Herpesvirus Humano 8 , Interações entre Hospedeiro e Microrganismos , Proteínas Imediatamente Precoces , Ubiquitina-Proteína Ligases , Proteínas Virais , Ativação Viral , Latência Viral , Replicação Viral , Humanos , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Ligação Proteica , Proteômica , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismoRESUMO
IMPORTANCE: A correlation exists between stress and increased episodes of human alpha-herpes virus 1 reactivation from latency. Stress increases corticosteroid levels; consequently, the glucocorticoid receptor (GR) is activated. Recent studies concluded that a GR agonist, but not an antagonist, accelerates productive infection and reactivation from latency. Furthermore, GR and certain stress-induced transcription factors cooperatively transactivate promoters that drive the expression of infected cell protein 0 (ICP0), ICP4, and VP16. This study revealed female mice expressing a GR containing a serine to alanine mutation at position 229 (GRS229A) shed significantly lower levels of infectious virus during explant-induced reactivation compared to male GRS229A or wild-type parental C57BL/6 mice. Furthermore, female GRS229A mice contained fewer VP16 + TG neurons compared to male GRS229A mice or wild-type mice during the early stages of explant-induced reactivation from latency. Collectively, these studies revealed that GR transcriptional activity has female-specific effects, whereas male mice can compensate for the loss of GR transcriptional activation.