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1.
Proc Natl Acad Sci U S A ; 121(5): e2308776121, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38252831

RESUMO

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.


Assuntos
Influenza Humana , Vírus , Animais , Camundongos , Humanos , Proteoma , Peptídeos/farmacologia , Descoberta de Drogas
2.
Int J Mol Sci ; 23(2)2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35055105

RESUMO

Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication.


Assuntos
Glucoquinase/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glicogênio/metabolismo , Glicólise , Interações Hospedeiro-Patógeno , Humanos , Metabolismo dos Lipídeos , Lipogênese , Mitocôndrias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química
3.
Commun Biol ; 4(1): 217, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594203

RESUMO

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK+/HK2- cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.


Assuntos
Metabolismo Energético , Glucoquinase/metabolismo , Hexoquinase/metabolismo , Imunidade Inata , Lipogênese , Neoplasias Hepáticas/enzimologia , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glucoquinase/genética , Hexoquinase/genética , Humanos , Isoenzimas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Transdução de Sinais
4.
J Immunol ; 201(5): 1510-1521, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30037846

RESUMO

Cell metabolism now appears as an essential regulator of immune cells activation. In particular, TLR stimulation triggers metabolic reprogramming of dendritic cells (DCs) with an increased glycolytic flux, whereas inhibition of glycolysis alters their functional activation. The molecular mechanisms involved in the control of glycolysis upon TLR stimulation are poorly understood for human DCs. TLR4 activation of human monocyte-derived DCs (MoDCs) stimulated glycolysis with an increased glucose consumption and lactate production. Global hexokinase (HK) activity, controlling the initial rate-limiting step of glycolysis, was also increased. TLR4-induced glycolytic burst correlated with a differential modulation of HK isoenzymes. LPS strongly enhanced the expression of HK2, whereas HK3 was reduced, HK1 remained unchanged, and HK4 was not expressed. Expression of the other rate-limiting glycolytic enzymes was not significantly increased. Exploring the signaling pathways involved in LPS-induced glycolysis with various specific inhibitors, we observed that only the inhibitors of p38-MAPK (SB203580) and of HIF-1α DNA binding (echinomycin) reduced both the glycolytic activity and production of cytokines triggered by TLR4 stimulation. In addition, LPS-induced HK2 expression required p38-MAPK-dependent HIF-1α accumulation and transcriptional activity. TLR1/2 and TLR2/6 stimulation increased glucose consumption by MoDCs through alternate mechanisms that are independent of p38-MAPK activation. TBK1 contributed to glycolysis regulation when DCs were stimulated via TLR2/6. Therefore, our results indicate that TLR4-dependent upregulation of glycolysis in human MoDCs involves a p38-MAPK-dependent HIF-1α accumulation, leading to an increased HK activity supported by enhanced HK2 expression.


Assuntos
Células Dendríticas/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Hexoquinase/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Monócitos/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Células Cultivadas , Células Dendríticas/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/toxicidade , Monócitos/patologia , Estabilidade Proteica , Receptor 4 Toll-Like/agonistas
5.
Sci Rep ; 7: 40791, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28106157

RESUMO

Dysregulated Toll-like receptor (TLR)-4 activation is involved in acute systemic sepsis, chronic inflammatory diseases, such as atherosclerosis and diabetes, and in viral infections, such as influenza infection. Thus, therapeutic control of the TLR4 signalling pathway is of major interest. Here we tested the activity of the small-molecule synthetic TLR4 antagonist, FP7, in vitro on human monocytes and monocyte-derived dendritic cells (DCs) and in vivo during influenza virus infection of mice. Our results indicate that FP7 antagonized the secretion of proinflammatory cytokines (IL-6, IL-8, and MIP-1ß) by monocytes and DCs (IC50 < 1 µM) and prevented DC maturation upon TLR4 activation by ultrapure lipopolysaccharide (LPS). FP7 selectively blocked TLR4 stimulation, but not TLR1/2, TLR2/6, or TLR3 activation. TLR4 stimulation of human DCs resulted in increased glycolytic activity that was also antagonized by FP7. FP7 protected mice from influenza virus-induced lethality and reduced both proinflammatory cytokine gene expression in the lungs and acute lung injury (ALI). Therefore, FP7 can antagonize TLR4 activation in vitro and protect mice from severe influenza infection, most likely by reducing TLR4-dependent cytokine storm mediated by damage-associated molecular patterns (DAMPs) like HMGB1.


Assuntos
Citocinas/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Vírus da Influenza A/imunologia , Lipopolissacarídeos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Citocinas/genética , Células Dendríticas/citologia , Relação Dose-Resposta a Droga , Feminino , Glucose/metabolismo , Glicólise , Mediadores da Inflamação , Masculino , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Monossacarídeos/farmacologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo
6.
J Virol ; 89(16): 8462-73, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26041289

RESUMO

UNLABELLED: Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope. IMPORTANCE: JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transformação Celular Viral/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/fisiologia , Adenomatose Pulmonar Ovina/fisiopatologia , Doenças dos Ovinos/fisiopatologia , Doenças dos Ovinos/virologia , Animais , Western Blotting , Primers do DNA/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Imunoprecipitação , Complexos Multiproteicos/metabolismo , Fases de Leitura Aberta/genética , RNA Interferente Pequeno/genética , Ovinos , Estatísticas não Paramétricas , Técnicas do Sistema de Duplo-Híbrido
7.
EMBO Rep ; 14(10): 938-44, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24008843

RESUMO

Virus-host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication.


Assuntos
Interações Hospedeiro-Patógeno , Modelos Biológicos , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cães , Humanos , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Orthomyxoviridae/metabolismo , Orthomyxoviridae/fisiologia , Ligação Proteica , Proteínas não Estruturais Virais/química , Replicação Viral
8.
PLoS Pathog ; 9(7): e1003440, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23853584

RESUMO

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.


Assuntos
Adenosina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Replicação Viral , Adenosina Desaminase/química , Adenosina Desaminase/genética , Transporte Biológico , Linhagem Celular , Vírus da Dengue/enzimologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade da Espécie , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética
9.
Curr Opin Virol ; 2(5): 606-13, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23025912

RESUMO

Viruses are recurrent socio economical and health problems each year worldwide. Current drugs are mainly directed against viral components and select resistant strains that urge the need to develop new antiviral therapeutics. High-throughput screening technologies now allow to draw comprehensive genome-wide maps of physical and genetic virus-host interactions. This has been done recently for several viruses such as HIV, HCV, DENV and FLUAV and revealed a wealth of potential antiviral cellular targets. Systems-level analysis of virus-host protein networks and subnetworks begins to uncover several specific points of intervention for a human centered drug development. We present here this new paradigm in antiviral drug discovery together with the first promising antiviral molecules.


Assuntos
Antivirais/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Viroses/metabolismo , Vírus/efeitos dos fármacos , Animais , Descoberta de Drogas , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Viroses/genética , Viroses/virologia , Vírus/genética , Vírus/metabolismo
10.
Mol Biosyst ; 8(4): 1297-303, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22307679

RESUMO

Current anti-influenza virus drugs target two viral proteins and induce a selective pressure for the generation of drug resistant variants. This stresses the need for additional therapeutic strategies including drug targeting of cellular factors that are essential for viral replication. Reverse genetics approaches can be used to identify these factors and recently six independent genomic initiatives have led to the identification of 925 host factors that are essential for the replication of influenza viruses. Here we report a meta-analysis of this dataset, first revealing that these screens are poorly overlapping at the gene level. However, a strong convergence was observed at the level of biological processes which was further supported by an interactomic analysis showing a high interconnectivity of the essential host factors in the human protein network. Plugging virus-host protein interaction data on this dataset reveals a significant targeting of these factors by viral proteins, further validating the cellular targets. Combining this information, the first drug-influenza virus target network was constructed by retrieving from DrugBank 298 molecules interacting with 100 essential host factors. Of these, 204 are FDA-approved offering interesting potential for rapid drug repositioning in the treatment of flu.


Assuntos
Descoberta de Drogas , Orthomyxoviridae/fisiologia , Genética Reversa/métodos , Antivirais/farmacologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Orthomyxoviridae/genética , Proteínas Virais/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
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