Endoplasmic Reticulum Quality Control Pathways Research Articles

FUT10 and FUT11 are protein O-fucosyltransferases that modify protein EMI domains.

O-Fucosylation plays crucial roles in various essential biological events. Alongside the well-established O-fucosylation of epidermal growth factor-like repeats by protein O-fucosyltransferase 1 (POFUT1) and thrombospondin type 1 repeats by POFUT2, we recently identified a type of O-fucosylation on the elastin microfibril interface (EMI) domain of Multimerin-1 (MMRN1). Here, using AlphaFold2 screens, co-immunoprecipitation, enzymatic assays combined with mass spectrometric analysis and CRISPR-Cas9 knockouts, we demonstrate that FUT10 and FUT11, originally annotated in UniProt as α1,3-fucosyltransferases, are actually POFUTs responsible for modifying EMI domains; thus, we renamed them as POFUT3 and POFUT4, respectively. Like POFUT1/2, POFUT3/4 function in the endoplasmic reticulum, require folded domain structures for modification and participate in a non-canonical endoplasmic reticulum quality control pathway for EMI domain-containing protein secretion. This finding expands the O-fucosylation repertoire and provides an entry point for further exploration in this emerging field of O-fucosylation.

DERL2 (derlin 2) stabilizes BAG6 (BAG cochaperone 6) in chemotherapy resistance of cholangiocarcinoma.

DERL2 (derlin 2) is a critical component of the endoplasmic reticulum quality control pathway system whose mutations play an important role in carcinogenesis, including cholangiocarcinoma (CHOL). However, its role and its underlying mechanism have yet to be elucidated. Herein, we revealed that DERL2 was highly expressed in CHOL and considered as an independent prognostic indicator for inferior survival in CHOL. DERL2 ectopically expressed in CHOL cells promoted cell proliferation and colony formation rates, and depleting DERL2 in CHOL cells curbed tumor growth in vitro and in vivo. More interestingly, the knockout of DERL2 augmented the growth-inhibitory effect of gemcitabine chemotherapy on CHOL cells by inducing cell apoptosis. Mechanistically, we discovered that DERL2 interacted with BAG6 (BAG cochaperone 6), thereby extending its half-life and reinforcing the oncogenic role of BAG6 in CHOL progression.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 μg ml−1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

Silencing of the proteasome and oxidative stress impair endoplasmic reticulum targeting and signal cleavage of a prostate carcinoma antigen

The endoplasmic reticulum (ER) is an organelle with high protein density and therefore prone to be damaged by protein aggregates. One proposed preventive measure is a pre-emptive quality control pathway that attenuates ER import during protein folding stress. ER resident proteins are targeted into the ER via signal peptides cleaved rapidly upon ER insertion by the ER signal peptidase. Here we show that the ER insertion and cleavage of the ER-targeting peptide of the prostate carcinoma antigen prostate stem cell antigen (PSCA) is retarded and strongly reduced when the proteasome is inhibited or genetically silenced. Also overexpression of the C-terminally extended ubiquitin variant Ub2-UBB+1 or oxidative stress attenuated signal peptide processing. Proteasome inhibition likewise protracted ER signal processing of the ER targeted hormone leptin and the MHC class I molecule H–2Dd. These findings, which are consistent with a pre-emptive ER quality control pathway, may explain why an immunodominant MHC class I peptide ligand of PSCA spanning its ER signal peptidase cleavage site is efficiently generated in the cytoplasm from PSCA precursors that fail to reach the ER.

Cleavage and degradation of EDEM1 promotes coxsackievirus B3 replication via ATF6a-mediated unfolded protein response signalling.

Our previous study of coxsackievirus B3 (CVB3)-induced unfolded protein responses (UPR) found that overexpression of ATF6a enhances CVB3 VP1 capsid protein production and increases viral particle formation. These findings implicate that ATF6a signalling benefits CVB3 replication. However, the mechanism by which ATF6a signalling is transduced to promote virus replication is unclear. In this study, using a Tet-On inducible ATF6a HeLa cell line, we found that ATF6a signalling downregulated the protein expression of the endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like protein 1 (EDEM1), resulting in accumulation of CVB3 VP1 protein; in contrast, expression of a dominant negative ATF6a had the opposite effect. Furthermore, we found that EDEM1 was cleaved by both CVB3 protease 3C and virus-activated caspase and subsequently degraded via the ubiquitin-proteasome pathway. However, overexpression of EDEM1 caused VP1 degradation, likely via a glycosylation-independent and ubiquitin-lysosome pathway. Finally, we demonstrated that CRISPR/Cas9-mediated knockout of EDEM1 increased VP1 accumulation and thus CVB3 replication. This is the first study to report the ER protein quality control of non-enveloped RNA virus and reveals a novel mechanism by which CVB3 evades host ER quality control pathways through cleavage and degradation of the UPR target gene EDEM1, to ultimately benefit its own replication.

Analyzing the Effects of O-Fucosylation on Secretion of ADAMTS Proteins Using Cell-Based Assays.

Metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 repeats) superfamily are extensively modified with glycan moieties. Glycosylation occurs as these enzymes are trafficked through the endoplasmic reticulum (ER) and Golgi apparatus on their way to the extracellular space and includes N-linked glycosylation, O-linked fucosylation and C-linked mannosylation. This chapter focuses on O-linked fucose, which is added to properly folded thrombospondin type 1 repeats (TSRs) in the ER by protein O-fucosyltransferase 2 (POFUT2) and elongated to a Glucoseβ1-3Fucose disaccharide by β3-glucosyltransferase (B3GLCT). Knockout of POFUT2 results in embryonic lethality in mice, and inactivating mutations in B3GLCT cause Peters plus syndrome, a congenital disorder of glycosylation in humans. Addition of the disaccharide by POFUT2 and B3GLCT stabilizes folded TSRs, enhancing folding in the ER and secretion efficiency of several ADAMTS proteins from cells. Thus, POFUT2 and B3GLCT both function as an ER quality control pathway for folding of TSRs in ADAMTS proteins. In this chapter we describe in detail the methods developed to analyze secretion defects of ADAMTS proteins upon loss of either POFUT2 or B3GLCT. The methods described include creation of CRISPR/Cas9-mediated knockout cell lines of POFUT2 and B3GLCT and use of these cell lines to analyze and quantify secretion defects of ADAMTS proteins.

Schwann cells ER-associated degradation contributes to myelin maintenance in adult nerves and limits demyelination in CMT1B mice.

In the peripheral nervous system (PNS) myelinating Schwann cells synthesize large amounts of myelin protein zero (P0) glycoprotein, an abundant component of peripheral nerve myelin. In humans, mutations in P0 cause the demyelinating Charcot-Marie-Tooth 1B (CMT1B) neuropathy, one of the most diffused genetic disorders of the PNS. We previously showed that several mutations, such as the deletion of serine 63 (P0-S63del), result in misfolding and accumulation of P0 in the endoplasmic reticulum (ER), with activation of the unfolded protein response (UPR). In addition, we observed that S63del mouse nerves display the upregulation of many ER-associated degradation (ERAD) genes, suggesting a possible involvement of this pathway in the clearance of the mutant P0. In ERAD in fact, misfolded proteins are dislocated from the ER and targeted for proteasomal degradation. Taking advantage of inducible cells that express the ER retained P0, here we show that the P0-S63del glycoprotein is degraded via ERAD. Moreover, we provide strong evidence that the Schwann cell-specific ablation of the ERAD factor Derlin-2 in S63del nerves exacerbates both the myelin defects and the UPR in vivo, unveiling a protective role for ERAD in CMT1B neuropathy. We also found that lack of Derlin-2 affects adult myelin maintenance in normal nerves, without compromising their development, pinpointing ERAD as a previously unrecognized player in preserving Schwann cells homeostasis in adulthood. Finally, we provide evidence that treatment of S63del peripheral nerve cultures with N-Acetyl-D-Glucosamine (GlcNAc), known to enhance protein quality control pathways in C.elegans, ameliorates S63del nerve myelination ex vivo. Overall, our study suggests that potentiating adaptive ER quality control pathways might represent an appealing strategy to treat both conformational and age-related PNS disorders.

Endoplasmic reticulum stress inhibits expression of genes involved in thyroid hormone synthesis and their key transcriptional regulators in FRTL-5 thyrocytes.

Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins due to an impairment of ER quality control pathways leading to the activation of a defense system, called unfolded protein response (UPR). While thyrocytes are supposed to be highly susceptible to environmental conditions that cause ER stress due to the synthesis of large amounts of secretory proteins required for thyroid hormone synthesis, systematic investigations on the effect of ER stress on expression of key genes of thyroid hormone synthesis and their transcriptional regulators are lacking. Since the aim of the ER stress-induced UPR is to restore ER homeostasis and to facilitate cell survival through transient shutdown of ribosomal protein translation, we hypothesized that the expression of genes involved in thyroid hormone synthesis and their transcriptional regulators, all of which are not essential for cell survival, are down-regulated in thyrocytes during ER stress, while sterol regulatory element-binding proteins (SREBPs) are activated during ER stress in thyrocytes. Treatment of FRTL-5 thyrocytes with the ER stress inducer tunicamycin (TM) dose-dependently increased the mRNA and/or protein levels of known UPR target genes, stimulated phosphorylation of the ER stress sensor protein kinase RNA-like ER kinase (PERK) and of the PERK target protein eukaryotic initiation factor 2α (eIF2α) and caused splicing of the ER stress-sensitive transcription factor X-box binding protein (XBP-1) (P < 0.05). The mRNA levels and/or protein levels of genes involved in thyroid hormone synthesis, sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), their transcriptional regulators and thyrotropin (TSH) receptor and the uptake of Na125I were reduced at the highest concentration of TM tested (0.1 μg/mL; P < 0.05). Proteolytic activation of the SREBP-1c pathway was not observed in FRTL-5 cells treated with TM, whereas TM reduced proteolytic activation of the SREBP-2 pathway at 0.1 μg TM/mL (P < 0.05). In conclusion, the expression of key genes involved in thyroid hormone synthesis and their critical regulators and of the TSH receptor as well as the uptake of iodide is attenuated in thyrocytes during mild ER stress. Down-regulation of NIS, TPO and TG during ER stress is likely the consequence of impaired TSH/TSHR signaling in concert with reduced expression of critical transcriptional regulators of these genes.

Altered fucosyltransferase expression in the superior temporal gyrus of elderly patients with schizophrenia

Glycosylation is a post-translational modification that is an essential element in cell signaling and neurodevelopmental pathway regulation. Glycan attachment can influence the tertiary structure and molecular interactions of glycosylated substrates, adding an additional layer of regulatory complexity to functional mechanisms underlying central cell biological processes. One type of enzyme-mediated glycan attachment, fucosylation, can mediate glycoprotein and glycolipid cell surface expression, trafficking, secretion, and quality control to modulate a variety of inter- and intracellular signaling cascades. Building on prior reports of glycosylation abnormalities and evidence of dysregulated glycosylation enzyme expression in schizophrenia, we examined the protein expression of 5 key fucose-modifying enzymes: GDP-fucose:protein O-fucosyltransferase 1 (POFUT1), GDP-fucose:protein O-fucosyltransferase 2 (POFUT2), fucosyltransferase 8 (FUT8), fucosyltransferase 11 (FUT11), and plasma α-l-fucosidase (FUCA2) in postmortem superior temporal gyrus of schizophrenia (N=16) and comparison (N=14) subjects. We also used the fucose binding protein, Aleuria aurantia lectin (AAL), to assess α-1,6-fucosylated N-glycoprotein abundance in the same subjects. In schizophrenia, we found increased expression of POFUT2, a fucosyltransferase uniquely responsible for O-fucosylation of thrombospondin-like repeat domains that is involved in a non-canonical endoplasmic reticulum quality control pathway. We also found decreased expression of FUT8 in schizophrenia. Given that FUT8 is the only α-1,6-fucosyltransferase expressed in mammals, the concurrent decrease in AAL binding in schizophrenia, particularly evident for N-glycoproteins in the ~52–58kDa and ~60–70kDa molecular mass ranges, likely reflects a consequence of abnormal FUT8 expression in the disorder. Dysregulated FUT8 and POFUT2 expression could potentially explain a variety of molecular abnormalities in schizophrenia.

A systems biology approach reveals new endoplasmic reticulum-associated targets for the correction of the ATP7B mutant causing Wilson disease

Copper (Cu) is an important trace element required for the activity of essential enzymes. However, excess Cu compromises the redox balance in cells and tissues causing serious toxicity. The process of disposal of excess Cu from organisms relies on the activity of Cu-transporting ATPase ATP7B. ATP7B is mainly expressed in liver hepatocytes where it sequesters the potentially toxic metal and mediates its excretion into the bile. Mutations in the ATP7B gene cause Wilson disease (WD), which is characterized by the accumulation of toxic Cu in the liver due to the scarce expression of ATP7B as well as the failure of ATP7B mutants to pump Cu and/or traffic to the Cu-excretion sites. The most frequent ATP7B mutant, H1069Q, still presents a significant Cu-transporting activity, but undergoes retention within the endoplasmic reticulum (ER) where the mutant is rapidly degraded. Expression of this ATP7B mutant has been recently reported to activate the p38 and JNK stress kinase pathways, which, in turn, trigger quality control mechanisms leading to the arrest of ATP7B-H1069Q in the ER and to the acceleration of its degradation. However, the main molecular players operating in these p38/JNK-dependent ER quality control pathways remain to be discovered. By using a combination of RNAseq, bioinformatics and RNAi approaches, we found a cluster of ER quality control genes whose expression is controlled by p38 and JNK and is required for the efficient retention of the ATP7B-H1069Q mutant in the ER. Silencing these genes reduced the accumulation of the ATP7B mutant in the ER and facilitated the mutant sorting and export to the Golgi and post-Golgi copper excretion sites. In sum, our findings reveal the ER-associated genes that could be utilized for the correction of ATP7B mutants and, hence, for the normalization of Cu homeostasis in Wilson disease.

Abstract C17: Revealing targetable cancer cell vulnerabilities by in silico pathway profiling

Abstract Successful targeted therapy of cancers relies on comprehensive understanding of vulnerabilities of the tumorigenic platform. Recent cancer genomics efforts have provided with an unprecedented opportunity to gain an unbiased insight into the functional make-up of human cancers. We have devised a computational strategy to leverage cancer genomic datasets to identify candidate pathway vulnerabilities in cancers. The core of our approach is in the delineation of molecular pathways that are activated in tumor cells as a result of secondary selection events during tumor evolution. These secondary pathways are likely to be required for tumorigenesis in the given tumor context. We applied this strategy on HER2+ breast cancers using breast cancer genomic data in the TCGA, the recently published METABRIC and the Cancer Cell Line Encyclopedia (CCLE) datasets. Although HER2+ breast cancers are treated with HER2-targeting agents in the clinic, the response is usually short-lived due to de novo and acquired resistance. We have found that HER2+ breast cancers are characterized by extensive and robust secondary upregulation of the endoplasmic reticulum (ER) quality control (ERQC) pathways, including the ER-associated degradation (ERAD) and PERK/eIF2 pathways. Our follow-up combined in silico and in vitro analyses on a panel of HER2+ and HER2- breast cancer cell lines revealed a model of HER2-driven tumorigenesis where yin-yang interplay between hyperactive HER2/mTOR signaling and the ERQC machinery regulate the ER protein homeostasis to control survival of HER2+ cells. Furthermore, we report that this delicate regulatory architecture can be exploited for therapeutic purposes, such that, combinatorial targeting of ERQC pathways to disturb the proteostatic balance synergized in selectively inducing proteotoxic cell death in therapy-refractory HER2+ cells. This study presents a computational method for identification of cancer pathway vulnerabilities, and suggests that exploiting proteotoxic ER stress may be an effective therapeutic strategy in therapy-refractory HER2+ breast cancers. Note: This abstract was not presented at the conference. Citation Format: Rashika Joshi, Kakajan Komurov. Revealing targetable cancer cell vulnerabilities by in silico pathway profiling. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C17.

A Bipartite Trigger for Dislocation Directs the Proteasomal Degradation of an Endoplasmic Reticulum Membrane Glycoprotein

Polypeptides are organized into distinct substructures, termed protein domains, that are often associated with diverse functions. These modular units can act as binding sites, areas of post-translational modification, and sites of complex multimerization. The human cytomegalovirus US2 gene product is organized into discrete domains that together catalyze the proteasome-dependent degradation of class I major histocompatibility complex heavy chains. US2 co-opts the endogenous ER quality control pathway in order to dispose of class I. The US2 endoplasmic reticulum (ER)-lumenal region is the class I binding domain, whereas the carboxyl terminus can be referred to as the degradation domain. In the present study, we examined the role of the US2 transmembrane domain in virus-mediated class I degradation. Replacement of the US2 transmembrane domain with that of the CD4 glycoprotein completely blocked the ability of US2 to induce class I destruction. A more precise mutagenesis revealed that subregions of the US2 transmembrane domain differ in their ability to trigger class I degradation. Collectively, the data support a model in which US2-mediated class I degradation occurs as a highly regulated process where the US2 transmembrane domain and cytoplasmic tail work in concert to eliminate class I molecules. Host factors, including a signal peptidase complex, probably associate with the US2 molecule in a coordinated fashion to create a predislocation complex to promote the extraction of class I out of the ER. The results imply that the ER quality control machinery may recognize and eliminate misfolded proteins using a similar multistep regulated process.

Lipid-mediated, reversible misfolding of a sterol-sensing domain protein

Cellular quality control requires recognition of common features of misfolding, and so is not typically associated with the specific targeting of individual proteins. However, physiologically regulated degradation of yeast HMG-CoA reductase (Hmg2p) occurs by the HRD endoplasmic reticulum quality control pathway, implying that Hmg2p undergoes a regulated transition to a quality control substrate in response to a sterol pathway molecule. Using in vitro structural assays, we now show that the pathway derivative farnesol causes Hmg2p to undergo a change to a less folded structure. The effect is reversible, biologically relevant by numerous criteria, highly specific for farnesol structure, and requires an intact Hmg2p sterol-sensing domain. This represents a distinct lipid-sensing function for this highly conserved motif that suggests novel approaches to cholesterol management. More generally, our observation of reversible small-molecule-mediated misfolding may herald numerous examples of regulated quality control to be discovered in biology or applied in the clinic.

Endoplasmic Reticulum (ER) Stress Induced by a Neurovirulent Mouse Retrovirus Is Associated with Prolonged BiP Binding and Retention of a Viral Protein in the ER

Some murine retroviruses cause a spongiform neurodegenerative disease exhibiting pathology resembling that observed in transmissible spongiform encephalopathies. The neurovirulence of these "spongiogenic retroviruses" is determined by the sequence of their respective envelope proteins, although the mechanisms of neurotoxicity are not understood. We have studied a highly neurovirulent virus called FrCasE that causes a rapidly progressive form of this disease. Recently, transcriptional markers of endoplasmic reticulum (ER) stress were detected during the early preclinical period in the brains of FrCasE-infected mice. In contrast, ER stress was not observed in mice infected with an avirulent virus, F43, which carries a different envelope gene, suggesting a role for ER stress in disease pathogenesis. Here we have examined in NIH 3T3 cells the cause of this cellular stress response. The envelope protein of F43 bound BiP, a major ER chaperone, transiently and was processed normally through the secretory pathway. In contrast, the envelope protein of FrCasE bound to BiP for a prolonged period, was retained in the ER, and was degraded by the proteasome. Furthermore, engagement of the FrCasE envelope protein by ER quality control pathways resulted in decreased steady-state levels of this protein, relative to that of F43, both in NIH 3T3 cells and in the brains of infected mice. Thus, the ER stress induced by FrCasE appears to be initiated by inefficient folding of its viral envelope protein, suggesting that the neurodegenerative disease caused by this virus represents a protein misfolding disorder.

Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae.

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6-166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.