ER-associated Degradation System Research Articles

Featured interactome of homocysteine-inducible endoplasmic reticulum protein uncovers novel binding partners in response to ER stress

Homocysteine-inducible endoplasmic reticulum protein (HERP) is an endoplasmic reticulum (ER)-resident protein and important for the adaptation of cellular protein homeostasis by ER-associated degradation (ERAD) system. HERP interactors are critical for cellular viability and the reaction to ER stress. To explore the exact mechanisms by which HERP performed the biological functions, we conducted an interaction analysis of HERP protein in HeLa cells by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometer (LC-MS)/MS coupled with label-free quantification (LFQ). Among the interactome results, 123 proteins significantly interacted with HERP, which leads to numerous biological processes including protein import into nucleus, ubiquitin-dependent ERAD pathway, negative regulation of apoptotic process, and protein transport from ER, along with multiple pathways including several diseases, protein processing in ER, fatty acid metabolism, and steroid biosynthesis. Furthermore, we selected several prey proteins from the interactome data and confirmed that HERP interacted with ancient ubiquitous protein 1 (AUP1), Fas-associated factor family member 2 (FAF2), tripartite motif containing 47 (TRIM47), acyl-CoA synthetase long-chain family member 3 (ACSL3), sequestosome 1 (SQSTM1), and poly(rC) binding protein 2 (PCBP2) by Co-IP and confocal microscopy experiments, respectively. Moreover, the expression and location of several interacted proteins were obviously altered in response to ER stress induced by Thapsigargin stimulation and Enterovirus 71 infection. In conclusion, our findings revealed that the vital proteins interacted with HERP to mediate signaling transduction, thus providing novel clues for the mechanisms of HERP associated with ERAD and metabolism in response to ER stress under physiological and pathological conditions.

The manipulation of cell signaling and host cell biology by cholera toxin

Vibrio cholerae colonizes the small intestine and releases cholera toxin into the extracellular space. The toxin binds to the apical surface of the epithelium, is internalized into the host endomembrane system, and escapes into the cytosol where it activates the stimulatory alpha subunit of the heterotrimeric G protein by ADP-ribosylation. This initiates a cAMP-dependent signaling pathway that stimulates chloride efflux into the gut, with diarrhea resulting from the accompanying osmotic movement of water into the intestinal lumen. G protein signaling is not the only host system manipulated by cholera toxin, however. Other cellular mechanisms and signaling pathways active in the intoxication process include endocytosis through lipid rafts, retrograde transport to the endoplasmic reticulum, the endoplasmic reticulum-associated degradation system for protein delivery to the cytosol, the unfolded protein response, and G protein de-activation through degradation or the function of ADP-ribosyl hydrolases. Although toxin-induced chloride efflux is thought to be an irreversible event, alterations to these processes could facilitate cellular recovery from intoxication. This review will highlight how cholera toxin exploits signaling pathways and other cell biology events to elicit a diarrheal response from the host.

Interview with the Guest Editor – James Olzmann

ABSTRACT James Olzmann did his undergraduate studies in biology at the University of Michigan, USA and subsequently obtained his PhD working on pathogenic mechanisms of DJ-1 in Parkinson's disease, under the supervision of Lian Li and Lih-Shen Chin at Emory University School of Medicine, Atlanta. For his postdoc, James joined the lab of Ron Kopito at Stanford University in 2007, where he studied different aspects of the ER-associated degradation system; through this research, he then became interested in and started working on lipid droplets. James set up his independent research group in 2013 at University of California Berkeley, and since 2019 he has been an Associate Professor. He is also an Investigator at the Chan Zuckerberg Biohub. His lab employs a combination of systems biology, chemical biology and cell biology strategies to understand the mechanisms that govern organelle and lipid homeostasis in health and disease. James is the Guest Editor for the 2022 Cell Biology of Lipids Special Issue in Journal of Cell Science.

Effects of flurbiprofen on the functional regulation of serotonin transporter and its misfolded mutant

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

Purification, Characterization, and Gene Expression of Rice Endo-β-N-Acetylglucosaminidase, Endo-Os.

In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-β-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-β-GlcNAc-ases and preliminarily reported the gene information of rice endo-β-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-β-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-β-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manβ1 unit; this substrate specificity was almost the same as that of other plant endo-β-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

Overexpression of ZmNF-YC13 Confers ER Stress Tolerance in Maize

Excessive accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER) can trigger ER stress. Although regulation of this stress response in plants is an important and effective means of mitigating the damage caused by stress, the associated genes and mechanisms in maize remain largely unknown. In this study, we identified the ER stress-responsive gene ZmNF-YC13, which encodes a protein localized in the nuclei and cytoplasm of maize cells. The overexpression of ZmNF-YC13 in maize and Arabidopsis was found to confer plant ER stress tolerance, and gene expression analyses revealed that the overexpression of this gene upregulates the expression of genes involved in protein folding and the ER-associated degradation (ERAD) system, including ERdj, BiP, and E3 ubiquitin ligase. Moreover, we established that ZmNF-YC13 activates the promoters of the chaperone genes ZmERdj and ZmBiP in protoplasts, which function in the elimination of unfolded and misfolded proteins in the ER. Based on our findings, we propose that ZmNF-YC13 positively regulates the ER stress response in maize by controlling chaperone genes, and we believe that these results could provide valuable information for further maize resistance breeding.

P97 Inhibitor CB-5083 Blocks ERAD in Trypanosoma brucei

Misfolded proteins trapped in the endoplasmic reticulum (ER) are specifically recognized and retrotranslocated to the cytosol by the ER-Associated Degradation (ERAD) system and delivered to the proteasome for destruction. This process was recently described in Trypanosoma brucei (T. brucei) using the misfolded epitope tagged Transferrin Receptor subunits ESAG7:Ty and HA:ESAG6 (HA:E6). Critical to this work was the proteasomal inhibitor MG132. However, MG132 has off-target inhibitory effects on lysosomal Cathepsin L that could cause misinterpretation of turnover results. Here, we evaluate an orally bioavailable p97 inhibitor, CB-5083, for use in T. brucei. p97 is a ubiquitous protein involved in many cellular events including the membrane extraction step of ERAD. CB-5083 strongly inhibits turnover of HA:E6, with comparable protein recovery to MG132 treatment. Interestingly, little deglycosylated cytoplasmic species accumulates, though it normally emerges with MG132 treatment. This suggests that CB-5083 blocks ERAD upstream of the proteasome, as expected for inhibition of the trypanosomal p97 orthologue TbVCP. Under CB-5083 treatment, HA:E6 is also strongly membrane-associated, suggesting ER localization. Finally, we provide an experimental example where CB-5083 treatment offers clarity to the off-target effects of MG132 treatment.

Effects of Maternal Fructose Intake on Perinatal ER-Stress: A Defective XBP1s Nuclear Translocation Affects the ER-stress Resolution.

Endoplasmic reticulum (ER) homeostasis is crucial to appropriate cell functioning, and when disturbed, a safeguard system called unfolded protein response (UPR) is activated. Fructose consumption modifies ER homeostasis and has been related to metabolic syndrome. However, fructose sweetened beverages intake is allowed during gestation. Therefore, we investigate whether maternal fructose intake affects the ER status and induces UPR. Thus, administrating liquid fructose (10% w/v) to pregnant rats partially activated the ER-stress in maternal and fetal liver and placenta. In fact, a fructose-induced increase in the levels of pIRE1 (phosphorylated inositol requiring enzyme-1) and its downstream effector, X-box binding protein-1 spliced form (XBP1s), was observed. XBP1s is a key transcription factor, however, XBP1s nuclear translocation and the expression of its target genes were reduced in the liver of the carbohydrate-fed mothers, and specifically diminished in the fetal liver and placenta in the fructose-fed mothers. These XBP1s target genes belong to the ER-associated protein degradation (ERAD) system, used to buffer ER-stress and to restore ER-homeostasis. It is known that XBP1s needs to form a complex with diverse proteins to migrate into the nucleus. Since methylglyoxal (MGO) content, a precursor of advanced glycation endproducts (AGE), was augmented in the three tissues in the fructose-fed mothers and has been related to interfere with the functioning of many proteins, the role of MGO in XBP1s migration should not be discarded. In conclusion, maternal fructose intake produces ER-stress, but without XBP1s nuclear migration. Therefore, a complete activation of UPR that would resolve ER-stress is lacking. A state of fructose-induced oxidative stress is probably involved.

A binding motif for Hsp90 in the A chains of ADP-ribosylating toxins that move from the endoplasmic reticulum to the cytosol.

Cholera toxin (Ctx) is an AB-type protein toxin that acts as an adenosine diphosphate (ADP)-ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER-associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N-terminal RPPDEI sequence (residues 11-16) and an LDIAPA sequence in the C-terminal region (residues 153-158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full-length CtxA1. Both sequences were necessary for the ER-to-cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI-like motif at the N- or C-termini of the A chains from four other ER-translocating toxins that act as ADP-ribosyltransferases: pertussis toxin, Escherichia coli heat-labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP-ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER-to-cytosol export of CtxA1 and possibly other toxin A chains as well.

Methods for genetic analysis of mammalian ER-associated degradation.

Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. We also discuss considerations for screen design, execution, and interpretation.

Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes

Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and β cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of β cells in females but not in males.

ER-localized protein-Herpud1 is a new mediator of IL-4-induced macrophage polarization and migration

ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response.

Rot1, an essential yeast protein, is degraded through the ER-associated protein degradation system (ERAD)

Rot1, an essential yeast protein, is degraded through the ER-associated protein degradation system (ERAD)

Melatonin reduces endoplasmic reticulum stress and corneal dystrophy‐associated TGFBIp through activation of endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER) stressis emerging as a factor forthepathogenesisof granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress-induced death than were wild-type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase-3 activation, and poly (ADP-ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin. Under ER stress, melatonin significantly suppressed the induction of immunoglobulin heavy-chain-binding protein (BiP) and activation of inositol-requiring enzyme 1α (IRE1α), and their downstream target, alternative splicing of X-box binding protein 1(XBP1). Notably, the reduction in BiP and IRE1α by melatonin was suppressed by the ubiquitin-proteasome inhibitor, MG132, but not by the autophagy inhibitor, bafilomycin A1, indicating involvement of the ER-associated protein degradation (ERAD) system. Melatonin treatment reduced the levels of transforming growth factor-β-induced protein (TGFBIp) significantly, and this reduction was suppressed by MG132. We also found reduced mRNA expression of theERADsystem components HRD1 and SEL1L, and a reduced level of SEL1L protein in GCD2 cells. Interestingly, melatonin treatments enhanced SEL1L levels and suppressed the inhibition of SEL1L N-glycosylation caused by tunicamycin. In conclusion, this study provides new insights into the mechanisms by which melatonin confers its protective actions during ER stress. The results also indicate that melatonin might have potential as a therapeutic agent for ER stress-related diseases including GCD2.

SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation.

Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.

The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins.

Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.

The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble proteins for degradation.

In eukaryotes, many proteins function in multi-subunit complexes that require proper assembly. To maintain complex stoichiometry, cells use the endoplasmic reticulum-associated degradation system to degrade unassembled membrane subunits, but how unassembled soluble proteins are eliminated is undefined. Here we show that degradation of unassembled soluble proteins (referred to as unassembled soluble protein degradation, USPD) requires the ubiquitin selective chaperone p97, its co-factor nuclear protein localization protein 4 (Npl4), and the proteasome. At the ubiquitin ligase level, the previously identified protein quality control ligase UBR1 (ubiquitin protein ligase E3 component n-recognin 1) and the related enzymes only process a subset of unassembled soluble proteins. We identify the homologous to the E6-AP carboxyl terminus (homologous to the E6-AP carboxyl terminus) domain-containing protein HUWE1 as a ubiquitin ligase for substrates bearing unshielded, hydrophobic segments. We used a stable isotope labeling with amino acids-based proteomic approach to identify endogenous HUWE1 substrates. Interestingly, many HUWE1 substrates form multi-protein complexes that function in the nucleus although HUWE1 itself is cytoplasmically localized. Inhibition of nuclear entry enhances HUWE1-mediated ubiquitination and degradation, suggesting that USPD occurs primarily in the cytoplasm. Altogether, these findings establish a new branch of the cytosolic protein quality control network, which removes surplus polypeptides to control protein homeostasis and nuclear complex assembly.

The molecular transportation across intracellular membrane system

Intracellular membranes constitute a complicate system which defines the cellular compartments, such as mitochondria, lysosome and endoplasmic reticulum (ER), and restricts biological active substrates within specific locations to conduct their physiological roles. Due to the hydrophobic property of the membranes, most of the enclosed molecules are unable to pass through the “lipidic walls” spontaneously and thus their traveling among different cell locations should be facilitated by the protein machinery across the intracellular membranes. In respect to eukaryotic cells, various transportation systems have evolved to orchestrate the complex traffic network connecting different cellular compartments in an efficient and organized way, including active transporter, passive transporter, channels, etc., which catalyze the transmembrane permeation of critical nutrient molecules, ions and even macromolecules. On the other hand, the lipidic environment within the membrane makes it a perfect place for biochemical reaction involving the hydrophobic molecules, such as the synthesis of steroids and sphingomyelins. The proper function of intracellular transportation systems is critical for cells to maintain a healthy metabolism, conduct their physiological activities in organism, and keep on the right track of cell fate, such as proliferation, differentiation and apoptosis. For example, the metabolism reactions in mitochondria require a sufficient supplies of biochemical substrates (e.g., pyruvate) to generate energy for cells, as well as a quick cleanup for the products and by products of the reaction to complete the cycles. The dysfunction of the systems could result in abnormal status of cell organelles: insufficient nutrition supplies would slow down the energy generation; excessive accumulation of certain molecules within mitochondria and ERs would be toxic and thus disturb the biological activities therein; incorrect traffic of proteins would cause their improper localization and lead to pathological process and diseases, such as cystic fibrosis resulting from the inappropriate localization of cystic fibrosis transmembrane conductance regulator (CFTR), and viral invasion by cytomegalovirus which can hijack the ER-associated degradation system to eliminate MHC I molecules by inducing MHC I to stray into cytoplasm instead of its proper destination, cell membrane, thus weakening the immune recognition of the cell. The research in the field of intracellular transportation runs far behind the progress of transportation across the cell membrane due to the difficulties in quantitation of the molecules inside cell compartments, live imaging on the dynamic change of cell organelle contents, and purification of membrane protein localized on intracellular membranes, and thus the breakthrough in the related technologies will prompt our understanding on the intracellular traffic greatly.

HRD1-mediated ERAD tuning of ER-bound E2 is conserved between plants and mammals

When membrane proteins and secretory proteins are misfolded or incompletely folded, they are retained in the endoplasmic reticulum (ER) for further folding or degradation. The HMG-COA reductase degradation 1 (HRD1) and degradation of alpha2 10 (DOA10) complexes are two major components involved in the ER-associated protein degradation (ERAD) system in eukaryotic organisms(1-4). However, the relationship between these two complexes is largely unknown, especially in higher eukaryotes. Here, we report that the plant ubiquitin-conjugating enzyme 32 (UBC32), an ER-bound E2 working in the DOA10 complex, is maintained at low levels under standard conditions by proteasome-dependent degradation mediated by the HRD1 complex, the other E3 complex involved in ERAD. Loss of this negative regulation under ER stress increases capacity for degradation of misfolded proteins retained in the ER. Consistently, UBE2J1, the homologue of UBC32 in mammals, was also identified to be targeted by HRD1 for degradation. Taken together, these results suggest that the regulation of UBC32 (or UBE2J1) by the HRD1 complex is conserved between plants and mammals.

Retromer and Rab2-dependent trafficking mediate PS1 degradation by proteasomes in endocytic disturbance.

Accumulating evidence suggests that endocytic pathway deficits are involved in Alzheimer's disease pathogenesis. Several reports show that endocytic disturbance affects β-amyloid peptide (Aβ) cleavage from β-amyloid precursor protein (APP). Presenilin-1 (PS1) is the catalytic core of the γ-secretase complex required for Aβ generation. Previously, we showed that aging induces endocytic disturbance, resulting in the accumulation of Aβ and APP in enlarged endosomes. It remains unclear, however, whether PS1 localization and function are affected with endocytic disturbance. Here, we report that in endocytic disturbance, PS1 is transported from endosomes to ER/Golgi compartments via retromer trafficking, and that PS1 interacts with vacuolar protein sorting-associated protein 35 both invitro and invivo. Moreover, PS1 is degraded by proteasomes via a Rab2-dependent trafficking pathway, only during endocytic disturbance. These findings suggest that PS1 levels and localization in endosomes are regulated by retromer trafficking and ER-associated degradation system, even if endocytic disturbance significantly induces the endosomal accumulation of APP and β-site APP-cleaving enzyme 1. Results of this study also suggest that retromer deficiency can affect PS1 localization in endosomes, where Aβ cleavage mainly occurs, possibly leading to enhanced Aβ pathology. We proposed the following mechanism for intracellular transport of presenilin-1 (PS1). When endosome/lysosome trafficking is disturbed, PS1 is transported from endosome to endoplasmic reticulum (ER)/Golgi compartments via retromer and Rab2-mediated trafficking, and then degraded by endoplasmic reticulum-associated degradation (ERAD). Perturbations in this trafficking can cause abnormal endosomal accumulation of PS1, and then may lead to exacerbated Aβ pathology. Cover Image for this issue: doi: 10.1111/jnc.13318.