Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.

Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Compartmentalization and Selective Tagging for Disposal of Misfolded Glycoproteins.

The ability of mammalian cells to correctly identify and degrade misfolded secretory proteins, most of them bearing N-glycans, is crucial for their correct function and survival. An inefficient disposal mechanism results in the accumulation of misfolded proteins and consequent endoplasmic reticulum (ER) stress. N-glycan processing creates a code that reveals the folding status of each molecule, enabling continued folding attempts or targeting of the doomed glycoprotein for disposal. We review here the main steps involved in the accurate processing of unfolded glycoproteins. We highlight recent data suggesting that the processing is not stochastic, but that there is selective accelerated glycan trimming on misfolded glycoprotein molecules.

Grp94 Protein Delivers γ-Aminobutyric Acid Type A (GABAA) Receptors to Hrd1 Protein-mediated Endoplasmic Reticulum-associated Degradation.

Proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multisubunit, multispan, integral membrane protein, even wild type subunits of GABAA receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Unassembled and misfolded subunits undergo ER-associated degradation (ERAD), but this degradation process remains poorly understood for GABAA receptors. Here, using the α1 subunits of GABAA receptors as a model substrate, we demonstrated that Grp94, a metazoan-specific Hsp90 in the ER lumen, uses its middle domain to interact with the α1 subunits and positively regulates their ERAD. OS-9, an ER-resident lectin, acts downstream of Grp94 to further recognize misfolded α1 subunits in a glycan-dependent manner. This delivers misfolded α1 subunits to the Hrd1-mediated ubiquitination and the valosin-containing protein-mediated extraction pathway. Repressing the initial ERAD recognition step by inhibiting Grp94 enhances the functional surface expression of misfolding-prone α1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies a Grp94-mediated ERAD pathway for GABAA receptors, which provides a novel way to finely tune their function in physiological and pathophysiological conditions.

Quality control of glycoprotein folding and ERAD: the role of N-glycan handling, EDEM1 and OS-9.

Protein N-glycosylation and quality control of protein folding as well as the connected ER-associated degradation of misfolded glycoproteins (ERAD) are not only evolutionary highly conserved but also functionally linked. It is now established that particular N-glycan structures which result from processing reactions by exo-glycosidases in the ER are of importance for glycoprotein folding and for ERAD. Thus, mono-glucosylated N-glycan intermediates harbor structural information which is important for promoting glycoprotein folding. On the other hand, specific mannose-trimmed N-glycans harbor structural information for routing misfolded glycoproteins to ERAD. In this review, we summarize current knowledge concerning the role played by glucosidases I and II, in concert with the bifunctional glucosyltransferase and calnexin/calreticulin in glycoprotein folding, the role of conventional ER mannosidase I in concert with the mannosidase EDEM1 in handling and routing of misfolded glycoproteins, and how the bifunctional OS-9 provides a link to the ER dislocon for degradation.

SEL1L degradation intermediates stimulate cytosolic aggregation of polyglutamine-expanded protein.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). In mammalian cells, the HRD1-SEL1L membrane ubiquitin ligase complex plays a central role in this process. However, SEL1L is inherently unstable, and excess SEL1L is also degraded by ERAD. Accordingly, when proteasome activity is inhibited, multiple degradation intermediates of SEL1L appear in the cytosol. In this study, we searched for factors that inhibit SEL1L degradation and identified OS-9 and XTP3-B, two ER lectins that regulate glycoprotein ERAD. SEL1L degradation was characterized by a ladder of degradation products, and the C-terminal Pro-rich region of SEL1L was responsible for generation of this pattern. In the cytosol, these degradation intermediates stimulated aggregation of polyglutamine-expanded Huntingtin protein (Htt-polyQ-GFP) by interacting with aggregation-prone proteins, including Htt-polyQ-GFP. Collectively, our findings indicate that peptide fragments of ER proteins generated during ERAD may affect protein aggregation in the cytosol, revealing the interconnection of protein homeostasis across subcellular compartments.

Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1.

BACKGROUND: Endoplasmic reticulum (ER)-associated degradation (ERAD) regulates protein homeostasis in the secretory pathway by targeting misfolded or unassembled proteins for degradation by the proteasome. Hrd1 is a conserved multi-spanning membrane bound ubiquitin ligase required for ubiquitination of many aberrant ER proteins, but few endogenous substrates of Hrd1 have been identified to date. METHODS: Using a SILAC-based quantitative proteomic approach combined with CRISPR-mediated gene silencing, we searched for endogenous physiological substrates of Hrd1. We used RNA microarray, immunoblotting, cycloheximide chase combined with chemical genetics to define the role of Hrd1 in regulating the stability of endogenous ERAD substrates. RESULTS: We identified 58 proteins whose levels are consistently upregulated in Hrd1 null HEK293 cells. Many of these proteins function in pathways involved in stress adaptation or immune surveillance. We validated OS9, a lectin required for ERAD of glycoproteins as a highly upregulated protein in Hrd1 deficient cells. Moreover, the abundance of OS9 is inversely correlated with Hrd1 level in clinical synovium samples isolated from osteoarthritis and rheumatoid arthritis patients. Intriguingly, immunoblotting detects two OS9 variants, both of which are upregulated when Hrd1 is inactivated. However, only one of these variants is subject to proteasome dependent degradation that requires Hrd1 and the AAA (ATPase associated with diverse cellular activities) ATPase p97. The stability of the other variant on the other hand is influenced by a lysosomal inhibitor. CONCLUSION: Hrd1 regulates the stability of proteins involved in ER stress response and immune activation by both proteasome dependent and independent mechanisms.

A Novel Role of OS-9 in the Maintenance of Intestinal Barrier Function from Hypoxia-induced Injury via p38-dependent Pathway.

OS-9 is a lectin required for efficient ubquitination of glycosylated substrates of endoplasmic reticulum-associated degradation (ERAD). OS-9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. However, we know very little about other functions of OS-9 under endoplasmic reticulum stress. Here, we used gene knockdown and overexpression approaches to study the protective effect of OS-9 on intestinal barrier function of intestinal epithelial cell Caco-2 monolayer. We found that OS-9 attenuated intestinal epithelial barrier dysfunction under hypoxia through up-regulating occludin and claudin-1 protein expression. Furthermore, we showed that the up-regulation of occludin and claudin-1 induced by OS-9 was mediated by p38 and ERK1/2 phosphorylation and did not involve HIF-1α. In summary, our results demonstrate that OS-9 up-regulates occludin and claudin-1 by activating the MAP kinase (MAPK) pathway, and thus protects the epithelial barrier function of Caco-2 monolayer under hypoxia condition.

Unconventional use of LC3 by coronaviruses through the alleged subversion of the ERAD tuning pathway.

Pathogens of bacterial and viral origin hijack pathways operating in eukaryotic cells in many ways in order to gain access into the host, to establish themselves and to eventually produce their progeny. The detailed molecular characterization of the subversion mechanisms devised by pathogens to infect host cells is crucial to generate targets for therapeutic intervention. Here we review recent data indicating that coronaviruses probably co-opt membranous carriers derived from the endoplasmic reticulum, which contain proteins that regulate disposal of misfolded polypeptides, for their replication. In addition, we also present models describing potential mechanisms that coronaviruses could employ for this hijacking.