Nobuko Hosokawa

A time-dependent phase shift in the mammalian unfolded protein response

Unfolded or misfolded proteins in the endoplasmic reticulum (ER) must be refolded or degraded to maintain homeostasis of the ER. The ATF6 and IRE1-XBP1 pathways are important for the refolding process in mammalian cells; activation of these transcriptional programs culminates in induction of ER-localized molecular chaperones and folding enzymes. We show here that degradation of misfolded glycoprotein substrates requires transcriptional induction of EDEM (ER degradation-enhancing alpha-mannosidase-like protein), and that this is mediated specifically by IRE1-XBP1 and not by ATF6. As XBP1 is produced after ATF6 activation, our results reveal a time-dependent transition in the mammalian unfolded protein response: an ATF6-mediated unidirectional phase (refolding only) is followed by an XBP1-mediated bidirectional phase (refolding plus degradation) as the response progresses.

A novel ER α-mannosidase-like protein accelerates ER-associated degradation

The quality control mechanism in the endoplasmic reticulum (ER) discriminates correctly folded proteins from misfolded polypeptides and determines their fate. Terminally misfolded proteins are retrotranslocated from the ER and degraded by cytoplasmic proteasomes, a mechanism known as ER‐associated degradation (ERAD). We report the cDNA cloning of Edem, a mouse gene encoding a putative type II ER transmembrane protein. Expression of Edem mRNA was induced by various types of ER stress. Although the luminal region of ER degradation enhancing α‐mannosidase‐like protein (EDEM) is similar to class I α1,2‐mannosidases involved in N‐glycan processing, EDEM did not have enzymatic activity. Overexpression of EDEM in human embryonic kidney 293 cells accelerated the degradation of misfolded α1‐antitrypsin, and EDEM bound to this misfolded glycoprotein. The results suggest that EDEM is directly involved in ERAD, and targets misfolded glycoproteins for degradation in an N‐glycan dependent manner.

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC 50 value was 32–55 μM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 μM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.

EDEM3, a Soluble EDEM Homolog, Enhances Glycoprotein Endoplasmic Reticulum-associated Degradation and Mannose Trimming

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing α-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I α-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded α1-antitrypsin variant (null (Hong Kong)) and of TCRα. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded α1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent α1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of α1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has α1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRDeltaF508, by specifically promoting ubiquitylation of CFTRDeltaF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRDeltaF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRDeltaF508 and catalyzes further polyubiquitylation of CFTRDeltaF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRDeltaF508.

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of α1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER α1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.

Human XTP3-B Forms an Endoplasmic Reticulum Quality Control Scaffold with the HRD1-SEL1L Ubiquitin Ligase Complex and BiP

The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human α1-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.

EDEM1 accelerates the trimming of α1,2-linked mannose on the C branch of N-glycans

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.

EDEM accelerates ERAD by preventing aberrant dimer formation of misfolded α1‐antitrypsin

Misfolded glycoproteins are degraded by a mechanism known as ERAD (ER‐associated degradation) after retrotranslocation out of the endoplasmic reticulum (ER). This mechanism plays an important role in ER quality control. We previously reported that an ER membrane protein, EDEM, accelerates ERAD of a misfolded α1‐antitrypsin variant, null (Hong Kong) (NHK), suggesting that EDEM may function as an acceptor of terminally misfolded glycoproteins. In this study, we constructed several genetically manipulated cell lines to test this hypothesis. EDEM expression did not alter the secretion rate of properly folded molecules and the forced retention of wild‐type α1‐antitrypsin in the ER did not cause its association with EDEM, suggesting that EDEM may function as a molecular chaperone. To examine this possibility, we analyzed the effect of EDEM over‐expression on the structure of NHK, and found that the accumulation of covalent NHK dimers was selectively prevented by the over‐expression of EDEM. Co‐expression of NHK with two other ER membrane proteins, calnexin and H+/K+‐ATPase (β subunit), did not inhibit NHK dimer formation or accelerate NHK ERAD. These results indicate that EDEM may maintain the retrotranslocation competence of NHK by inhibiting aggregation so that unstable misfolded proteins can be accommodated by the dislocon for ERAD.