Characterization of the Human Adenovirus 19a E3/49K Protein

Human adenoviruses (Ads) cause a variety of acute diseases but also establish persistent infections. Ads have evolved a number of mechanisms to evade the host immune response that seem to facilitate virus replication and transmission in vivo. Interestingly, one Ad transcription unit, the early transcription unit 3 (E3), appears to encode exclusively proteins with immuno-modulatory functions. Recently, a novel open reading frame, E3/49K, was identified in the E3 region of the epidemic keratoconjunctivitis causing serotype Ad19a. The sequence predicts a type I transmembrane protein with a large N-terminal domain and a short cytoplasmic tail of 19 amino acids (aa). The N-terminal part contains three repeat regions (R1-3) of about 80 aa each with R3 exhibiting a significant homology to immunoglobulin-like domains. This study shows that 49K is a highly glycosylated protein with an apparent molecular mass of 80-100 kDa. 12-13 out of 14 predicted N-glycosylation sites are utilized, and there is also evidence for O-glycosylation. 49K contains intramolecular disulfide bonds and is proteolytically processed. As a result of the cleavage the N-terminal ectodomain is secreted: Ad19a E3/49K is the first Ad protein known to be secreted. At steady-state, E3/49K is detected in the Golgi/TGN, early endosomes and at the cell surface of Ad19a-infected as well as 49K-transfected cells. The cytoplasmic tail, which contains two potential endosomal/lysosomal targeting motifs, YXXF and LL, plays a major role in the 49K trafficking. The proteolytic processing may occur in the TGN or secretory vesicles and/or at the cell surface. The finding that an increased cell surface expression of 49K correlates with an increased secretion rate indicates proteolytic processing of 49K at the cell surface. The C-terminal fragments and also unprocessed 49K proteins are internalized possibly by the interaction of the LL motif in the cytoplasmic tail with the clathrin adaptor complex AP-2. From early endosomes 49K proteins and C-terminal fragments recycle to the TGN potentially involving the interaction of the cytoplasmic tail of 49K with the clathrin adaptor complex AP-1 or to the cell surface, whereas a fraction is delivered to late endosomes and lysosomes. Remarkably, the secreted form of 49K specifically binds to various lymphocyte cell lines, including NK, B and T cell lines, but not to other cell types, e.g. fibroblasts, suggesting that the soluble and possibly also the membrane-bound 49K binds to receptor(s) on lymphocytes. Indeed, in Jurkat (T cell leukemia) cells, 49K-binding protein(s) with apparent molecular masses of 172-196 kDa were identified. Moreover, purified 49K inhibited the NK cell-mediated lysis of target cells. Thus, upon binding to cell surface receptors, 49K may trigger an inhibitory signal or inhibit an activating signal. While other Ad E3 proteins affect the infected cell, 49K seems to influence infiltrating cells of the immune system. Therefore, 49K function appears to represent an entirely novel Ad immune evasion mechanism

Kiri Chr. W. F. Walchile, Göttingen

http://tartu.ester.ee/record=b1881198~S1*es

Polyubiquitin binding to ABIN1 is required to prevent autoimmunity

The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor kappa B (NF-kappa B) essential modulator (NEMO), a component of the inhibitor of NF-kappa B (I kappa B) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88(-/-) mice, demonstrating that toll-like receptor (TLR)-MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-alpha/beta, c-Jun N-terminal kinases, and p38 alpha mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR-MyD88 pathways and prevent autoimmunity

Structural analysis of the adenovirus type 2 E3/19K protein using mutagenesis and a panel of conformation-sensitive monoclonal antibodies

The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide–MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3–13, 15–21 and 41–45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six β-strands and an α-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition

The primary cilium influences interleukin-1 beta-induced NF kappa B signalling by regulating IKK activity

AbstractThe primary cilium is an organelle acting as a master regulator of cellular signalling. We have previously shown that disruption of primary cilia assembly, through targeting intraflagellar transport, is associated with muted nitric oxide and prostaglandin responses to the inflammatory cytokine interleukin-1β (IL-1β). Here, we show that loss of the primary cilium disrupts specific molecular signalling events in cytosolic NFκB signalling. The induction of cyclooxygenase 2 (COX2) and inducible nitrous oxide synthase (iNOS) protein is abolished. Cells unable to assemble cilia exhibit unaffected activation of IκB kinase (IKK), but delayed and reduced degradation of IκB, due to diminished phosphorylation of inhibitor of kappa B (IκB) by IKK. This results in both delayed and reduced NFκB p65 nuclear translocation and nuclear transcript binding. We also demonstrate that heat shock protein 27 (hsp27), an established regulator of IKK, is localized to the ciliary axoneme and cellular levels are dramatically disrupted with loss of the primary cilium. These results suggest that the primary cilia compartment exerts influence over NFκB signalling. We propose that the cilium is a locality for regulation of the molecular events defining NFκB signalling events, tuning signalling as appropriate

Micro- and Nanoplastics’ Effects on Protein Folding and Amyloidosis

A significant portion of the world's plastic is not properly disposed of and, through various processes, is degraded into microscopic particles termed micro- and nanoplastics. Marine and terrestrial faunae, including humans, inevitably get in contact and may inhale and ingest these microscopic plastics which can deposit throughout the body, potentially altering cellular and molecular functions in the nervous and other systems. For instance, at the cellular level, studies in animal models have shown that plastic particles can cross the blood-brain barrier and interact with neurons, and thus affect cognition. At the molecular level, plastics may specifically influence the folding of proteins, induce the formation of aberrant amyloid proteins, and therefore potentially trigger the development of systemic and local amyloidosis. In this review, we discuss the general issue of plastic micro- and nanoparticle generation, with a focus on their effects on protein folding, misfolding, and their possible clinical implications

Interleukin-1β sequesters hypoxia inducible factor 2α to the primary cilium.

BACKGROUND: The primary cilium coordinates signalling in development, health and disease. Previously we have shown that the cilium is essential for the anabolic response to loading and the inflammatory response to interleukin-1β (IL-1β). We have also shown the primary cilium elongates in response to IL-1β exposure. Both anabolic phenotype and inflammatory pathology are proposed to be dependent on hypoxia-inducible factor 2 alpha (HIF-2α). The present study tests the hypothesis that an association exists between the primary cilium and HIFs in inflammatory signalling. RESULTS: Here we show, in articular chondrocytes, that IL-1β-induces primary cilia elongation with alterations to cilia trafficking of arl13b. This elongation is associated with a transient increase in HIF-2α expression and accumulation in the primary cilium. Prolyl hydroxylase inhibition results in primary cilia elongation also associated with accumulation of HIF-2α in the ciliary base and axoneme. This recruitment and the associated cilia elongation is not inhibited by blockade of HIFα transcription activity or rescue of basal HIF-2α expression. Hypomorphic mutation to intraflagellar transport protein IFT88 results in limited ciliogenesis. This is associated with increased HIF-2α expression and inhibited response to prolyl hydroxylase inhibition. CONCLUSIONS: These findings suggest that ciliary sequestration of HIF-2α provides negative regulation of HIF-2α expression and potentially activity. This study indicates, for the first time, that the primary cilium regulates HIF signalling during inflammation

Sorting motifs in the cytoplasmic tail of the immunomodulatory E3/49K protein of species D adenoviruses modulate cell surface expression and ectodomain shedding.

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved resulting in secretion of its large ectodomain (sec49K). Sec49K binds to CD45 on leukocytes, impairing activation and functions of NK cells and T cells. E3/49K is localized in the Golgi/trans-Golgi-network (TGN), early endosomes and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly-synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YxxΦ and LL that are important for binding the clathrin adaptor proteins AP-1 and AP-2 in vitro. Surprisingly, mutating the LL motif, either alone or together with YxxΦ, did not prevent proteolytic processing, but increased cell surface expression and secretion. Upon Brefeldin-A treatment cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix-metalloproteases

Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4

The E3 ubiquitin ligase Pellino can be activated by phosphorylation in vitro, catalyzed by IL-1 receptor-associated kinase 1 (IRAK1) or IRAK4. Here, we show that phosphorylation enhances the E3 ligase activity of Pellino 1 similarly with any of several E2-conjugating enzymes (Ubc13-Uev1a, UbcH4, or UbcH5a/5b) and identify 7 amino acid residues in Pellino 1 whose phosphorylation is critical for activation. Five of these sites are clustered between residues 76 and 86 (Ser-76, Ser-78, Thr-80, Ser-82, and Thr-86) and decorate a region of antiparallel β-sheet, termed the “wing,” which is an appendage of the forkhead-associated domain that is thought to interact with IRAK1. The other 2 sites are located at Thr-288 and Ser-293, just N-terminal to the RING-like domain that carries the E3 ligase activity. Unusually, the full activation of Pellino 1 can be achieved by phosphorylating any one of several different sites (Ser-76, Thr-86, Thr-288, or Ser-293) or a combination of other sites (Ser-78, Thr-80, and Ser-82). These observations imply that dephosphorylation of multiple sites is required to inactivate Pellino 1, which could be a device for prolonging Pellino's E3 ubiquitin ligase activity in vivo