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1.
ACS Chem Biol ; 17(6): 1315-1320, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35580266

RESUMO

Interleukin-1 receptor-associated kinase 3 (IRAK3) is a pseudokinase mediator in the human inflammatory pathway, and ablation of its function is associated with enhanced antitumor immunity. Traditionally, pseudokinases have eluded "druggability" and have not been considered tractable targets in the pharmaceutical industry. Herein we disclose a CRISPR/Cas9-mediated knockout of IRAK3 in monocyte-derived dendritic cells that results in an increase in IL-12 production upon lipopolysaccharide (LPS) stimulation. Furthermore, we disclose and characterize Degradomer D-1, which displays selective proteasomal degradation of IRAK3 and reproduces the 1L-12p40 increases observed in the CRISPR/Cas9 knockout.


Assuntos
Citocinas , Quinases Associadas a Receptores de Interleucina-1 , Citocinas/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-12/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo
2.
J Virol ; 88(20): 11811-24, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25100846

RESUMO

Human cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of cytomegaloviruses, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. The MCMV genome, like the genomes of other beta- and gammaherpesviruses, encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the M33 GPCR carried by MCMV is an activator of CREB, NF-κB, and phospholipase C-ß signaling pathways and has been implicated in aspects of pathogenesis in vivo, including persistence in the salivary glands of BALB/c mice. In this study, we used immunocompetent nonobese diabetic (NOD) and immunocompromised NOD-scid-gamma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus with an M33 deletion (ΔM33) can replicate in the salivary gland of immunocompromised animals, albeit with a 400-fold growth defect compared with the growth of wild-type virus. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the salivary gland. We conclude that the reduced replicative capacity of the ΔM33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since the salivary gland represents a site essential for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread. Importance: Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lives. Under a number of circumstances, the virus can reactivate, leading to a variety of diseases in both adults and developing babies, and therefore, identifying the function of viral proteins is essential to understand how the virus spreads and causes disease. We aim to utilize animal models to study the function of an important class of viral proteins termed G protein-coupled receptors with the ultimate goal of developing inhibitors to these proteins that could one day be used to prevent viral spread.


Assuntos
Citomegalovirus/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/genética , Glândulas Salivares/virologia , Animais , Linhagem Celular , Citomegalovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
J Virol ; 83(16): 8141-52, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19494016

RESUMO

The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric G(q/11) proteins and not through promiscuous coupling of M33 to the G(s) pathway. Using inhibitors of signaling molecules downstream of G(q/11), we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-beta and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38alpha and transcription factor NF-kappaB, occurs in the absence of G(q/11) and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-beta and PKC plays a central role in MCMV pathogenesis in vivo.


Assuntos
Infecções por Herpesviridae/metabolismo , Muromegalovirus/fisiologia , Fosfolipase C beta/metabolismo , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Feminino , Infecções por Herpesviridae/enzimologia , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Fosfolipase C beta/genética , Proteína Quinase C/genética , Receptores Acoplados a Proteínas G/genética , Glândulas Salivares/enzimologia , Glândulas Salivares/metabolismo , Glândulas Salivares/virologia , Proteínas Virais/genética , Replicação Viral
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