Respiratory syncytial virus NS1 protein degrades STAT2 by using the elongin-cullin E3 ligase

Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.

Prevalence of refractive error in Europe: the European Eye Epidemiology (E3) Consortium

To estimate the prevalence of refractive error in adults across Europe. Refractive data (mean spherical equivalent) collected between 1990 and 2013 from fifteen population-based cohort and cross-sectional studies of the European Eye Epidemiology (E³) Consortium were combined in a random effects meta-analysis stratified by 5-year age intervals and gender. Participants were excluded if they were identified as having had cataract surgery, retinal detachment, refractive surgery or other factors that might influence refraction. Estimates of refractive error prevalence were obtained including the following classifications: myopia ≤−0.75 diopters (D), high myopia ≤−6D, hyperopia ≥1D and astigmatism ≥1D. Meta-analysis of refractive error was performed for 61,946 individuals from fifteen studies with median age ranging from 44 to 81 and minimal ethnic variation (98 % European ancestry). The age-standardised prevalences (using the 2010 European Standard Population, limited to those ≥25 and <90 years old) were: myopia 30.6 % [95 % confidence interval (CI) 30.4–30.9], high myopia 2.7 % (95 % CI 2.69–2.73), hyperopia 25.2 % (95 % CI 25.0–25.4) and astigmatism 23.9 % (95 % CI 23.7–24.1). Age-specific estimates revealed a high prevalence of myopia in younger participants [47.2 % (CI 41.8–52.5) in 25–29 years-olds]. Refractive error affects just over a half of European adults. The greatest burden of refractive error is due to myopia, with high prevalence rates in young adults. Using the 2010 European population estimates, we estimate there are 227.2 million people with myopia across Europe.

Molecular and physiological roles of Pellino E3 ubiquitin ligases in immunity

The sensing of foreign agents by the innate and adaptive immune system triggers complex signal transduction cascades that culminate in expression of gene patterns that facilitate host protection from the invading agent. Post-translational modification of intracellular signaling proteins in these pathways is a key regulatory mechanism with ubiquitination being one of the important processes that controls levels and activities of signaling molecules. E3 ubiquitin ligases are the determining enzymes in dictating the ubiquitination status of individual proteins. Among these hundred E3 ubiquitin ligases are a family of Pellino proteins that are emerging to be important players in immunity and beyond. Herein, we review the roles of the Pellino E3 ubiquitin ligases in innate and adaptive immunity. We discuss their early discovery and characterization and how this has been aided by the highly conserved nature of innate immune signaling across evolution. We describe the molecular roles of Pellino proteins in immune signaling with particular emphasis on their involvement in pathogen recognition receptor (PRR) signaling. The growing appreciation of the importance of Pellino proteins in a wide range of immune-mediated diseases are also evaluated.

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.

The roles of Pellino E3 ubiquitin ligases in immunity

Pellino proteins were initially characterized as a family of E3 ubiquitin ligases that can catalyse the ubiquitylation of interleukin-1 receptor-associated kinase 1 (IRAK1) and regulate innate immune signalling pathways. More recently, physiological and molecular roles for members of the Pellino family have been described in the regulation of innate and adaptive immune responses by ubiquitylation. This Review describes the emerging roles of Pellino proteins in innate and adaptive immunity and discusses the mechanistic basis of these functions.

Ophthalmic epidemiology in Europe: the “European Eye Epidemiology” (E3) consortium

The European Eye Epidemiology (E3) consortium is a recently formed consortium of 29 groups from 12 European countries. It already comprises 21 population-based studies and 20 other studies (case-control, cases only, randomized trials), providing ophthalmological data on approximately 170,000 European participants. The aim of the consortium is to promote and sustain collaboration and sharing of data and knowledge in the field of ophthalmic epidemiology in Europe, with particular focus on the harmonization of methods for future research, estimation and projection of frequency and impact of visual outcomes in European populations (including temporal trends and European subregions), identification of risk factors and pathways for eye diseases (lifestyle, vascular and metabolic factors, genetics, epigenetics and biomarkers) and development and validation of prediction models for eye diseases. Coordinating these existing data will allow a detailed study of the risk factors and consequences of eye diseases and visual impairment, including study of international geographical variation which is not possible in individual studies. It is expected that collaborative work on these existing data will provide additional knowledge, despite the fact that the risk factors and the methods for collecting them differ somewhat among the participating studies. Most studies also include biobanks of various biological samples, which will enable identification of biomarkers to detect and predict occurrence and progression of eye diseases. This article outlines the rationale of the consortium, its design and presents a summary of the methodology.

Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils

An early and critical event in beta2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent beta2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) beta2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3´,5´-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected beta2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in beta2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.

SOCS2 Can Enhance Interleukin-2 (IL-2) and IL-3 Signalling by Accelerating SOCS3 Degradation

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.

CD33 reponses are blocked by SOCS3 through accelerated proteasomal-mediated turnover

CD33 is a member of the sialic acid–binding immunoglobulin-like lectin (Siglec) family of inhibitory receptors and a therapeutic target for acute myeloid leukemia (AML). CD33 contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which can recruit SHP-1 and SHP-2. How CD33 expression is regulated is unclear. Suppressor of cytokine signaling 3 (SOCS3) is expressed in response to cytokines, LPS, and other PAMPs, and competes with SHP-1/2 binding to ITIMs of cytokine receptors, thereby inhibiting signaling. In this study, using peptide pull-down experiments, we found that SOCS3 can specifically bind to the phosphorylated ITIM of CD33. Additionally, following cross-linking SOCS3 can recruit the ECS E3 ligase resulting in accelerated proteasomal degradation of both CD33 and SOCS3. Our data suggest that the tyrosine motifs in CD33 are not important for internalization, while they are required for degradation. Moreover, SOCS3 inhibited the CD33-induced block on cytokine-induced proliferation. This is the first receptor shown to be degraded by SOCS3 and where SOCS3 and its target protein are degraded concomitantly. Our findings clearly suggest that during an inflammatory response, the inhibitory receptor CD33 is lost by this mechanism. Moreover, this has important clinical implications as tumors expressing SOCS3 may be refractory to -CD33 therapy.

SOCS3 Targets Siglec 7 for Proteasomal Degradation and Blocks Siglec 7-mediated Responses

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5–11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation

The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3'-OH kinase/protein kinase B (PKB) Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser(166) and Ser(188). Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser(166) and Ser(188) in a phosphatidylinositide 3'-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3'-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser(166) and Ser(188) and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBalpha displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21(Cip1), resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2.p53 signaling pathway by regulating Mdm2 stability.