ER-associated Degradation Pathway Research Articles

The Functional Classification of ORF8 in SARS-CoV-2 Replication, Immune Evasion, and Viral Pathogenesis Inferred through Phylogenetic Profiling.

ORF8 is a highly variable genomic region of SARS-CoV-2. Although non-essential and the precise functions are unknown, it has been suggested that this protein assists in SARS-CoV-2 replication in the early secretory pathway and in immune evasion. We utilized the binding partners of SARS-CoV-2 proteins in human HEK293T cells and performed genome-wide phylogenetic profiling and clustering analyses in 446 eukaryotic species to predict and discover ORF8 binding partners that share associated functional mechanisms based on co-evolution. Results classified 47 ORF8 binding partner proteins into 3 clusters (groups 1-3), which were conserved in vertebrates (group 1), metazoan (group 2), and eukaryotes (group 3). Gene ontology analysis indicated that group 1 had no significant associated biological processes, while groups 2 and 3 were associated with glycoprotein biosynthesis process and ubiquitin-dependent endoplasmic reticulum-associated degradation pathways, respectively. Collectively, our results classified potential genes that might be associated with SARS-CoV-2 viral pathogenesis, specifically related to acute respiratory distress syndrome, and the secretory pathway. Here, we discuss the possible role of ORF8 in viral pathogenesis and in assisting viral replication and immune evasion via secretory pathway, as well as the possible factors associated with the rapid evolution of ORF8.

Suppression of retinal degeneration by two novel ERAD ubiquitin E3 ligases SORDD1/2 in Drosophila.

Mutations in the gene rhodopsin are one of the major causes of autosomal dominant retinitis pigmentosa (adRP). Mutant forms of Rhodopsin frequently accumulate in the endoplasmic reticulum (ER), cause ER stress, and trigger photoreceptor cell degeneration. Here, we performed a genome-wide screen to identify suppressors of retinal degeneration in a Drosophila model of adRP, carrying a point mutation in the major rhodopsin, Rh1 (Rh1G69D). We identified two novel E3 ubiquitin ligases SORDD1 and SORDD2 that effectively suppressed Rh1G69D-induced photoreceptor dysfunction and retinal degeneration. SORDD1/2 promoted the ubiquitination and degradation of Rh1G69D through VCP (valosin containing protein) and independent of processes reliant on the HRD1 (HMG-CoA reductase degradation protein 1)/HRD3 complex. We further demonstrate that SORDD1/2 and HRD1 function in parallel and in a redundant fashion to maintain rhodopsin homeostasis and integrity of photoreceptor cells. These findings identify a new ER-associated protein degradation (ERAD) pathway and suggest that facilitating SORDD1/2 function may be a therapeutic strategy to treat adRP.

C-terminal tail length guides insertion and assembly of membrane proteins

A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

Suppressed ER-associated degradation by intraglomerular cross talk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease.

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. In vitro experiments revealed the suppression of the ER-associated degradation (ERAD) pathway and induction of apoptosis in podocytes that were stimulated with the supernatant of MCs cultured in high glucose conditions. In diabetic mice, ERAD inhibition resulted in exacerbated albuminuria, increased apoptosis in podocytes, and reduced nephrin expression associated with the downregulation of ERAD-related biomolecules. Flow cytometry analysis of podocytes isolated from MafB (a transcription factor known to be expressed in macrophages and podocytes)-GFP knock-in mice revealed that ERAD inhibition resulted in decreased nephrin phosphorylation. These findings suggest that an intraglomerular cross talk between MCs and podocytes can inhibit physiological ERAD processes and suppress the phosphorylation of nephrin in podocytes, which thereby lead to podocyte injury under diabetic conditions. Therapeutic intervention of the ERAD pathway through the cross talk between these cells is potentially a novel strategy for DKD.

ERADicating stem cells from their niche.

Protein homeostasis preserves stem cell function, but the underlying mechanisms are largely unknown. A study reveals that protein quality control mediated by the endoplasmic reticulum-associated degradation pathway ensures proper expression of MPL, a key cell surface receptor that promotes haematopoietic stem cell function through niche interaction.

Aneuploidy-induced proteotoxic stress can be effectively tolerated without dosage compensation, genetic mutations, or stress responses

BackgroundThe protein homeostasis (proteostasis) network maintains balanced protein synthesis, folding, transport, and degradation within a cell. Failure to maintain proteostasis is associated with aging and disease, leading to concerted efforts to study how the network responds to various proteotoxic stresses. This is often accomplished using ectopic overexpression of well-characterized, model misfolded protein substrates. However, how cells tolerate large-scale, diverse burden to the proteostasis network is not understood. Aneuploidy, the state of imbalanced chromosome content, adversely affects the proteostasis network by dysregulating the expression of hundreds of proteins simultaneously. Using aneuploid haploid yeast cells as a model, we address whether cells can tolerate large-scale, diverse challenges to the proteostasis network.ResultsHere we characterize several aneuploid Saccharomyces cerevisiae strains isolated from a collection of stable, randomly generated yeast aneuploid cells. These strains exhibit robust growth and resistance to multiple drugs which induce various forms of proteotoxic stress. Whole genome re-sequencing of the strains revealed this was not the result of genetic mutations, and transcriptome profiling combined with ribosome footprinting showed that genes are expressed and translated in accordance to chromosome copy number. In some strains, various facets of the proteostasis network are mildly upregulated without chronic activation of environmental stress response or heat shock response pathways. No severe defects were observed in the degradation of misfolded proteins, using model misfolded substrates of endoplasmic reticulum-associated degradation or cytosolic quality control pathways, and protein biosynthesis capacity was not impaired.ConclusionsWe show that yeast strains of some karyotypes in the genetic background studied here can tolerate the large aneuploidy-associated burden to the proteostasis machinery without genetic changes, dosage compensation, or activation of canonical stress response pathways. We suggest that proteotoxic stress, while common, is not always an obligate consequence of aneuploidy, but rather certain karyotypes and genetic backgrounds may be able to tolerate the excess protein burden placed on the protein homeostasis machinery. This may help clarify how cancer cells are paradoxically both highly aneuploid and highly proliferative at the same time.

Immunopeptidomic Analysis Reveals That Deamidated HLA-bound Peptides Arise Predominantly from Deglycosylated Precursors

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Ubiquitination of disease-causing CFTR variants in a microsome-based assay.

Soluble secreted proteins and membrane proteins are subjected to protein quality control pathways during their synthesis in the endoplasmic reticulum (ER) and delivery to other destinations. Foremost among these quality control pathways is the selection of misfolded proteins for ER-associated degradation (ERAD). A growing number of diseases, including Cystic Fibrosis, are linked to the ERAD pathway. In most cases, a membrane protein known as the Cystic Fibrosis Transmembrane Conductance Regulator, or CFTR, is prematurely degraded by ERAD. Cell-based assays and in vitro studies have elucidated factors required for the recognition and degradation of CFTR, yet mechanistic details on how these factors target specific disease-causing variants is limited. Given the possibility that variants might exhibit unique susceptibilities to ubiquitin modification, which is required for proteasome-mediated degradation, we devised an assay that recapitulates this event. Here, we demonstrate that ER-enriched membranes from transfected human cells support CFTR ubiquitination when combined with radiolabeled ubiquitin and isolated enzymes in the ubiquitination cascade. We also show that select disease-causing variants are ubiquitinated more extensively than wild-type channels and to varying degrees. Our system provides a platform to examine how other purified factors impact CFTR ubiquitination and the ubiquitination of additional disease-associated membrane proteins.

ER-associated Protein Degradation at Atomic Resolution

The endoplasmic reticulum-associated degradation (ERAD) pathway eliminates misfolded proteins. The Hrd1 complex represents the main gate mediating retrotranslocation of ER luminal misfolded (ERAD-L) substrates to the cytosol. A recent cryo-electron microscopy (cryo-EM) study by Wu et al. unveils the structural features of active Hrd1, providing mechanistic insights into the movement of proteins directed for degradation across ER membranes.

FoxO suppresses endoplasmic reticulum stress to inhibit growth of Tsc1-deficient tissues under nutrient restriction.

The transcription factor FoxO has been shown to block proliferation and progression in mTORC1-driven tumorigenesis but the picture of the relevant FoxO target genes remains incomplete. Here, we employed RNA-seq profiling on single clones isolated using laser capture microdissection from Drosophila larval eye imaginal discs to identify FoxO targets that restrict the proliferation of Tsc1-deficient cells under nutrient restriction (NR). Transcriptomics analysis revealed downregulation of endoplasmic reticulum-associated protein degradation pathway components upon foxo knockdown. Induction of ER stress pharmacologically or by suppression of other ER stress response pathway components led to an enhanced overgrowth of Tsc1 knockdown tissue. Increase of ER stress in Tsc1 loss-of-function cells upon foxo knockdown was also confirmed by elevated expression levels of known ER stress markers. These results highlight the role of FoxO in limiting ER stress to regulate Tsc1 mutant overgrowth.

Arabidopsis Ubiquitin-Conjugating Enzymes UBC7, UBC13, and UBC14 Are Required in Plant Responses to Multiple Stress Conditions.

Protein ubiquitination plays important roles in plants, including stress responses. The ubiquitin (Ub) E2 enzymes are required in the transfer of Ub to a substrate and are also important in determining the Ub-chain linkage specificity. However, for many of the 37 E2 genes in Arabidopsis thaliana, there is currently little or no understanding of their functions. In this study, we investigated three members of an E2 subfamily. The single, double, and triple mutants of UBC7, UBC13, and UBC14 did not show any phenotypic changes under normal conditions, but were more sensitive than the wild-type (WT) plants to multiple stress conditions, suggesting that the three genes are not critical for normal growth, but required in plant stress responses. The severity of the phenotypes increased from single to triple mutants, suggesting that the functions of the three genes are not completely redundant. The three E2s are closely related to the yeast Ubc7 and its homologs in animals and human, which are an important component of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The stress sensitivity phenotypes of the mutants and shared evolutionary root with the Ubc7 homologs in yeast and metazoans suggest that UBC7, UBC13, and UBC14 may function in the plant ERAD pathway.

Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)–protein disulfide-isomerase relay when protein folding capacity is exceeded

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD). The UPR is triggered by unfolded protein stress and leads to up-regulation of cellular components such as chaperones and folding catalysts. These components relieve stress by increasing folding capacity and up-regulating ERAD components that remove non-native proteins. Although oxidative protein folding and the UPR/ERAD pathways each are well-understood, very little is known about any direct cross-talk between them. In this study, we carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfide-isomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation–reduction cycles and may also enhance ERAD, which requires reduced protein substrates. Relief from excess levels of non-native proteins by increasing the levels of folding factors removed the feedback inhibition. These results reveal regulatory cross-talk between the oxidative protein folding and UPR and ERAD pathways.

49-OR: Synergistic Effect of ERAD and Autophagy in Preventing Proinsulin Aggregation and Promoting Beta-Cell Survival

Decline of proteostasis in the endoplasmic reticulum (ER) is thought to underlie molecular changes in β cell function and drive the pathogenesis of diabetes. ER-associated degradation (ERAD) and macroautophagy (hereafter “autophagy”) are two principal quality-control mechanisms responsible for the clearance of ER-resident proteins or protein aggregates, respectively, in the cell. However, the physiological significance of ERAD-autophagy crosstalk remains unexplored. Previously, using β cell-specific knockout mouse models, we reported that ERAD deficiency leads to β cell dedifferentiation, while autophagy deficiency leads to β cell loss by 4-5 months of age. Interestingly, we observed that loss of ERAD in β cells triggered activation of autophagy pathways, as monitored by LC3 flux. Furthermore, chloroquine-mediated inhibition of autophagy in ERAD-deficient islets led to an increased proinsulin aggregation. In mice, combined deletion of ERAD and autophagy in β cells caused marked hyperglycemia soon after weaning, with extensive loss of β cells and premature death at 2-3 months of age. Thus, our data demonstrate that ERAD and autophagy act synergistically to prevent proinsulin aggregation and promote β cell survival. This study reveals a critical role for crosstalk between the ERAD and autophagy pathways in preventing diabetes and provides the first evidence for synergism between ERAD and autophagy in vivo. Disclosure N. Shrestha: None. R.B. Reinert: None. P. Arvan: None. L. Qi: None. Funding JDRF (2-SRA-2018-539-A-B); National Institutes of Health (1R01DK117639-01, 1R01DK111174)

Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor.

SND1 is highly expressed in various cancers. Here, we identify oncoprotein SND1 as a previously unidentified endoplasmic reticulum (ER) membrane-associated protein. The amino-terminal peptide of SND1 predominantly associates with SEC61A, which anchors on ER membrane. The SN domain of SND1 catches and guides the nascent synthesized heavy chain (HC) of MHC-I to ER-associated degradation (ERAD), hindering the normal assembly of MHC-I in the ER lumen. In mice model bearing tumors, especially in transgenic OT-I mice, deletion of SND1 promotes the presentation of MHC-I in both B16F10 and MC38 cells, and the infiltration of CD8+ T cells is notably increased in tumor tissue. It was further confirmed that SND1 impaired tumor antigen presentation to cytotoxic CD8+ T cells both in vivo and in vitro. These findings reveal SND1 as a novel ER-associated protein facilitating immune evasion of tumor cells through redirecting HC to ERAD pathway that consequently interrupts antigen presentation.

Effect of Praziquantel on Schistosoma mekongi Proteome and Phosphoproteome.

Schistosoma mekongi causes schistosomiasis in southeast Asia, against which praziquantel (PZQ) is the only treatment option. PZQ resistance has been reported, thus increasing the requirement to understand mechanism of PZQ. Herein, this study aimed to assess differences in proteome and phosphoproteome of S. mekongi after PZQ treatment for elucidating its action. Furthermore, key kinases related to PZQ effects were predicted to identify alternative targets for novel drug development. Proteomes of S. mekongi were profiled after PZQ treatment at half maximal inhibitory concentration and compared with untreated worms. A total of 144 proteins were differentially expressed after treatment. In parallel, immunohistochemistry indicated a reduction of phosphorylation, with 43 phosphoproteins showing reduced phosphorylation, as identified by phosphoproteomic approach. Pathway analysis of mass spectrometric data showed that calcium homeostasis, worm antigen, and oxidative stress pathways were influenced by PZQ treatment. Interestingly, two novel mechanisms related to protein folding and proteolysis through endoplasmic reticulum-associated degradation pathways were indicated as a parasiticidal mechanism of PZQ. According to kinase–substrate predictions with bioinformatic tools, Src kinase was highlighted as the major kinase related to the alteration of phosphorylation by PZQ. Interfering with these pathways or applying Src kinase inhibitors could be alternative approaches for further antischistosomal drug development.

Pharmacologic inhibition of N-linked glycan trimming with kifunensine disrupts GLUT1 trafficking and glucose uptake

The facilitative glucose transport GLUT1 (SLC2A1) is a constitutively expressed membrane protein involved in basal uptake of blood glucose. GLUT1 modification by N-linked glycosylation at a single asparagine residue (N45) appears to play multiple roles in the trafficking, stability and transport activity of this protein. Here we examine the role of complex N-glycosylation on GLUT1 function in renal epithelial cells by arresting this modification at the high-mannose stage with the mannosidase I inhibitor kifunensine. Consistent with prior work in which GLUT1 glycosylation was completely inhibited, we find that kifunensine treatment results in a time-dependent decrease of up to 40% in cellular glucose uptake. We further demonstrate that this effect is primarily a result of deficient GLUT1 trafficking to the cell membrane due to quality control mechanisms that instead direct GLUT1 to the ER-associated degradation (ERAD) pathway. Unlike tunicamycin, which inhibits the first step in N-glycosyl transfer and causes dramatic cell cycle arrest, kifunensine causes only a modest decrease in GLUT1 levels and cell cycle progression in both normal and transformed renal cells. The effect of kifunensine on the cell cycle appears to be independent of its effect on GLUT1, since all renal cell types in this study displayed decreased proliferation regardless of their dependence on glucose uptake for growth and survival. Together these results indicate that proper N-glycan processing plays an important role in directing GLUT1 to the cell surface and that disruption of mannosidase activity results in aberrant degradation of GLUT1 by the ERAD pathway.

Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy.

Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that is widely used in industry as a host system for heterologous protein expression. Heterologous gene expression is typically facilitated by strongly inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters. However, protein production is usually accomplished by a fed-batch induction process, which is known to negatively affect cell physiology, resulting in limited protein yields and quality. To assess how yields of exogenous proteins can be increased and to further understand the physiological response of P. pastoris to the carbon conversion of glycerol and methanol, as well as the continuous induction of methanol, we analyzed recombinant protein production in a 10,000-L fed-batch culture. Furthermore, we investigated gene expression during the yeast cell culture phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth and may regulate the alcohol oxidase1 (AOX1) promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris heterologous protein expression, regulation, and secretory mechanisms.

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation.

The endoplasmic reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosaccharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalysed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. RHBDL4 thereby controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.

The yeast protein Ubx4p contributes to mitochondrial respiration and lithium–galactose–mediated activation of the unfolded protein response

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

Characterization of the CsPNG1 gene from cucumber and its function in response to salinity stress

Peptide: N-glycanase (PNGase; EC 3.5.1.52) is a deglycosylation enzyme that is responsible for deglycosylating misfolded glycoproteins in the endoplasmic reticulum. However, the role of PNGase in plants is largely unknown. Here, we cloned and characterized the function of peptide: N-glycanase (CsPNG1) from cucumber. The amino acid encoded by CsPNG1 gene contained a typical transglutaminase (TGase) catalytic triad domain and belonged to the “TGase superfamily”. Subcellular localization showed that CsPNG1 was located in the cell membrane and nucleus. Promoter sequence analysis and qPCR tests showed that CsPNG1 could respond to a variety of abiotic stresses and hormone treatments. Yeast one-hybrid assays revealed the interaction between the transcription factor CsGT-3b and CsPNG1 promoter. Importantly, overexpression of CsPNG1 in tobacco increased the tolerance to salt stress of transgenic plants. In addition, CsPNG1 interacted with CsRAD23 family proteins and the C-terminal UBA domain of CsRAD23 protein was responsible for binding to CsPNG1, indicating that CsPNG1 was involved in the ER-associated degradation pathway (ERAD). Taken together, our study demonstrated that CsPNG1 plays a positive role in improving plant salt tolerance, and these findings might provide a basis for further functional analysis of CsPNG1 genes in abiotic stress and ERAD.