Dampening of ISGylation of RIG-I by ADAP regulates type I interferon response of macrophages to RNA virus infection.

While macrophage is one of the major type I interferon (IFN-I) producers in multiple tissues during viral infections, it also serves as an important target cell for many RNA viruses. However, the regulatory mechanism for the IFN-I response of macrophages to respond to a viral challenge is not fully understood. Here we report ADAP, an immune adaptor protein, is indispensable for the induction of the IFN-I response of macrophages to RNA virus infections via an inhibition of the conjugation of ubiquitin-like ISG15 (ISGylation) to RIG-I. Loss of ADAP increases RNA virus replication in macrophages, accompanied with a decrease in LPS-induced IFN-ß and ISG15 mRNA expression and an impairment in the RNA virus-induced phosphorylation of IRF3 and TBK1. Moreover, using Adap-/- mice, we show ADAP deficiency strongly increases the susceptibility of macrophages to RNA-virus infection in vivo. Mechanically, ADAP selectively interacts and functionally cooperates with RIG-I but not MDA5 in the activation of IFN-ß transcription. Loss of ADAP results in an enhancement of ISGylation of RIG-I, whereas overexpression of ADAP exhibits the opposite effect in vitro, indicating ADAP is detrimental to the RNA virus-induced ISGylation of RIG-I. Together, our data demonstrate a novel antagonistic activity of ADAP in the cell-intrinsic control of RIG-I ISGylation, which is indispensable for initiating and sustaining the IFN-I response of macrophages to RNA virus infections and replication.

ADAP Y571 Phosphorylation Is Required to Prime STAT3 for Activation in TLR4-Stimulated Macrophages.

Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS. Furthermore, overexpression of ADAP enhances, whereas loss of ADAP reduces, the LPS-mediated phosphorylation and activity of STAT3, suggesting ADAP acts as a coactivator of STAT3 activity and function. Furthermore, the coactivator function of ADAP mostly depends on the tyrosine phosphorylation at Y571 in the motif YDSL induced by LPS. Mutation of Y571 to F severely impairs the stimulating effect of ADAP on STAT3 activity and the ability of ADAP to inhibit M1-like polarization in TLR4-activated mouse macrophages. Moreover, ADAP interacts with STAT3, and loss of ADAP renders mouse macrophages less sensitive to IL-6 stimulation for STAT3 phosphorylation. Collectively, our findings revealed an additional layer of regulation of TLR4-mediated mouse macrophage plasticity whereby ADAP phosphorylation on Y571 is required to prime STAT3 for activation in TLR4-stimulated mouse macrophages.

Endometrial stromal cell proteomic analysis reveals LIM and SH3 protein 1 (LASP1) plays important roles in the progression of adenomyosis.

Adenomyosis is one of the most common gynecological disorders that the molecular events underlying its pathogenesis remain not fully understood. Prior studies have shown that endometrial stromal cells (ESCs) played crucial roles in the pathogenesis of adenomyosis. In this study, we utilized two-dimensional gel electrophoresis combined with protein identification by mass spectrometry (2D/MS) proteomics analysis to compare the differential protein expression profile between the paired eutopic and ectopic ESCs (EuESCs and EcESCs) in adenomyosis, and a total of 32 significantly altered protein spots were identified. Among which, the expression of LIM and SH3 protein 1 (LASP1) was increased significantly in EcESCs compared to EuESCs. Immunohistochemical assay showed that LASP1 was overexpressed in the stromal cells of ectopic endometriums compared to eutopic endometriums; further functional analyses revealed that LASP1 overexpression could enhance cell proliferation, migration and invasion of EcESCs. Furthermore, we also showed that the dysregulated expression of LASP1 in EcESCs was associated with DNA hypermethylation in the promoter region of the LASP1 gene. However, the detailed molecular mechanisms of enhancing cell proliferation, invasion and migration caused by upregulated LASP1 in adenomyosis needs further study. For the first time, our data suggested that LASP1 plays important roles in the pathogenesis of adenomyosis, and could serve as a prognostic biomarker of adenomyosis.

Ubc9 Interacts with and SUMOylates the TCR Adaptor SLP-76 for NFAT Transcription in T Cells.

Although the immune adaptor SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) integrates and propagates the TCR signaling, the regulation of SLP-76 during the TCR signaling is incompletely studied. In this article, we report that SLP-76 interacts with the small ubiquitin-like modifier (SUMO) E2 conjugase Ubc9 and is a substrate for Ubc9-mediated SUMOylation in human and mouse T cells. TCR stimulation promotes SLP-76-Ubc9 binding, accompanied by an increase in SLP-76 SUMOylation. Ubc9 binds to the extreme C terminus of SLP-76 spanning residues 516-533 and SUMOylates SLP-76 at two conserved residues K266 and K284. In addition, SLP-76 and Ubc9 synergizes to augment the TCR-mediated IL-2 transcription by NFAT in a manner dependent of SUMOylation of SLP-76. Moreover, although not affecting the TCR proximal signaling events, the Ubc9-mediated SUMOylation of SLP-76 is required for TCR-induced assembly of Ubc9-NFAT complex for IL-2 transcription. Together, these results suggest that Ubc9 modulates the function of SLP-76 in T cell activation both by direct interaction and by SUMOylation of SLP-76 and that the Ubc9-SLP-76 module acts as a novel regulatory complex in the control of T cell activation.

Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli.

BACKGROUND: Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production. RESULT: In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate. CONCLUSIONS: The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt.

E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3.

Ubiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.