Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine (pTyr) sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its Nmyristoylation. Multiple receptor tyrosine kinases (RTKs) can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome integrity and activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies.
Classical swine fever virus (CSFV) is a highly contagious pathogen, which pose continuous threat to the swine industry. Though most attenuated vaccines are effective, they fail to serologically distinguish between infected and vaccinated animals, hindering CSFV eradication. Beneficially, nanoparticles (NPs)-based vaccines resemble natural viruses in size and antigen structure, and offer an alternative tool to circumvent these limitations. Using self-assembling NPs as multimerization platforms provides a safe and immunogenic tool against infectious diseases. This study presented a novel strategy to display CSFV E2 glycoprotein on the surface of genetically engineered self-assembling NPs. Eukaryotic E2-fused protein (SP-E2-mi3) could self-assemble into uniform NPs as indicated in transmission electron microscope (TEM) and dynamic light scattering (DLS). SP-E2-mi3 NPs showed high stability at room temperature. This NP-based immunization resulted in enhanced antigen uptake and up-regulated production of immunostimulatory cytokines in antigen presenting cells (APCs). Moreover, the protective efficacy of SP-E2-mi3 NPs was evaluated in pigs. SP-E2-mi3 NPs significantly improved both humoral and cellular immunity, especially as indicated by the elevated CSFV-specific IFN-γ cellular immunity and >10-fold neutralizing antibodies as compared to monomeric E2. These observations were consistent to in vivo protection against CSFV lethal virus challenge in prime-boost immunization schedule. Further results revealed single dose of 10 μg of SP-E2-mi3 NPs provided considerable clinical protection against lethal virus challenge. In conclusion, these findings demonstrated that this NP-based technology has potential to enhance the potency of subunit vaccine, paving ways for nanovaccine development.
PRRSV is a major swine pathogen, suppresses innate immunity, and causes persistent infection and coinfection with other pathogens. As a central immune mediator, MALT1 plays essential roles in regulating immunity and inflammation.
Monocyte chemotactic protein-induced protein 1 (MCPIP1) is an inflammatory regulator in immune response and has broad antiviral effects by targeting viral RNA. Porcine reproductive and respiratory syndrome virus (PRRSV), a major viral pathogen in pigs, causes immune suppression leading to co-infection of swine pathogens but the mechanisms are not fully clarified. In this study, MCPIP1 expression was found to be significantly up-regulated in lungs of PRRSV-infected piglets, as well as in Marc-145 and PAM cells upon PRRSV stimulation. MCPIP1 overexpression significantly inhibited PRRSV replication while MCPIP1 knock-down increased virus titer. Various mutations in RNase functional domains of MCPIP1 impaired the inhibitory activity against PRRSV, while those in deubiquitinase domains failed to. MCPIP1 expression started to decrease from 60 h post PRRSV infection in PAMs. Meanwhile, infection with higher dose of PRRSV further down-regulated MCPIP1, indicating the antagonizing effects from PRRSV against MCPIP1. Moreover, it was confirmed that MCPIP1 expression was down-regulated in 3D4 cells with either IL-17 or nsp11 overexpression, while IL-17 inhibitor abolished the decrease of MCPIP1 caused by nsp11, indicating nsp11 employs IL-17 induction to inhibit MCPIP1. Furthermore, PRRSV nsp11 mutant with deficiency in IL-17 induction showed the recovered expression of MCPIP1 in infected cells, inspiring a strategy for virus attenuation. This is the first report about the role of MCPIP1 against PRRSV and the function of PRRSV nsp11 against innate immunity to facilitate virus replication via IL-17. The study not only illuminates PRRSV infection machinery but also enlightens alternative antiviral strategies, such as vaccine candidates. Importance Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate immunity and leads to co-infection of swine pathogens. Monocyte chemotactic protein-induced protein 1 (MCPIP1) is a broad-spectrum host antiviral protein. Therefore, to further clarify the mechanism of PRRSV against innate immunity, we explored the relationship between MCPIP1 and PRRSV infection. The results showed that MCPIP1 inhibited PRRSV infection in the early stage of virus infection. Importantly, PRRSV nsp11 subsequently employed IL-17 induction to suppress MCPIP1 expression and antagonized anti-PRRSV effects. Furthermore, PRRSV with mutation of nsp11 S74A failed to induce MCPIP1 reduction. These findings confirmed the function of MCPIP1 against swine viruses and revealed that PRRSV nsp11 plays an important role in virus against innate immunity. This study enlightens a new strategy to develop safer attenuated vaccines against PRRSV by nsp11 mutation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.