Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation.

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

An endoplasmic reticulum-associated degradation-related E2-E3 enzyme pair controls grain size and weight through the brassinosteroid signaling pathway in rice.

Grain size is an important agronomic trait, but our knowledge about grain size determination in crops is still limited. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a special ubiquitin proteasome system that is involved in degrading misfolded or incompletely folded proteins in the ER. Here, we report that SMALL GRAIN 3 (SMG3) and DECREASED GRAIN SIZE 1 (DGS1), an ERAD-related E2-E3 enzyme pair, regulate grain size and weight through the brassinosteroid (BR) signaling pathway in rice (Oryza sativa). SMG3 encodes a homolog of Arabidopsis (Arabidopsis thaliana) UBIQUITIN CONJUGATING ENZYME 32, which is a conserved ERAD-associated E2 ubiquitin conjugating enzyme. SMG3 interacts with another grain size regulator, DGS1. Loss of function of SMG3 or DGS1 results in small grains, while overexpression of SMG3 or DGS1 leads to long grains. Further analyses showed that DGS1 is an active E3 ubiquitin ligase and colocates with SMG3 in the ER. SMG3 and DGS1 are involved in BR signaling. DGS1 ubiquitinates the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and affects its accumulation. Genetic analysis suggests that SMG3, DGS1, and BRI1 act together to regulate grain size and weight. In summary, our findings identify an ERAD-related E2-E3 pair that regulates grain size and weight, which gives insight into the function of ERAD in grain size control and BR signaling.

Lipid biosynthesis perturbation impairs endoplasmic reticulum-associated degradation.

The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.

A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation.

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.

Ngly1 -/- rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems.

N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.

Comparative proteomics reveals elevated CCN2 in NGLY1-deficient cells.

N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-ß signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-ß pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-ß signaling to microscopic liver fibrosis in NGLY1 patients.

A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61-2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61-2 grow at 25 °C because the protein is stable, but sec61-2 yeast are inviable at 38 °C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61-2 and hence sec61-2hrd1∆ double mutants are viable at 38 °C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1's transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.

Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice.

Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF). Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-ß1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses. Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-ß1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein. Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.

The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation.

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including MS-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

ERAD deficiency promotes mitochondrial dysfunction and transcriptional rewiring in human hepatic cells.

Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

Arabidopsis OTU1, a linkage-specific deubiquitinase, is required for endoplasmic reticulum-associated protein degradation.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is part of the ER protein quality-control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N-terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N-terminal region. These observations collectively define AtOTU1 as an OTU domain-containing deubiquitinase involved in Arabidopsis ERAD.

Endoplasmic Reticulum (ER) and ER-Phagy.

The endoplasmic reticulum (ER) is a biosynthetic organelle in eukaryotic cells. Its capacity to produce proteins, lipids and oligosaccharides responds to physiologic and pathologic demand. The transcriptional and translational unfolded protein response (UPR) programs increase ER size and activity. In contrast, ER-phagy programs in all their flavors select ER subdomains for lysosomal clearance. These programs are activated by nutrient deprivation, accumulation of excess ER (recov-ER-phagy), production of misfolded proteins that cannot be degraded by ER-associated degradation and that are removed from cells by the so-called ER-to-lysosome-associated degradation (ERLAD). Selection of ER subdomains to be cleared from cells relies on ER-phagy receptors, a class of membrane-bound proteins displaying cytosolic domains that engage the cytosolic ubiquitin-like protein LC3. Mechanistically, ER clearance proceeds via macro-ER-phagy, micro-ER-phagy and LC3-regulated vesicular delivery.

Defects in Protein Folding and/or Quality Control Cause Protein Aggregation in the Endoplasmic Reticulum.

Protein aggregation is now a common hallmark of numerous human diseases, most of which involve cytosolic aggregates including Aß (AD) and ⍺-synuclein (PD) in Alzheimer's disease and Parkinson's disease. However, it is also evident that protein aggregation can also occur in the lumen of the endoplasmic reticulum (ER) that leads to specific diseases due to loss of protein function or detrimental effects on the host cell, the former is inherited in a recessive manner where the latter are dominantly inherited. However, the mechanisms of protein aggregation, disaggregation and degradation in the ER are not well understood. Here we provide an overview of factors that cause protein aggregation in the ER and how the ER handles aggregated proteins. Protein aggregation in the ER can result from intrinsic properties of the protein (hydrophobic residues in the ER), oxidative stress or nutrient depletion. The ER has quality control mechanisms [chaperone functions, ER-associated protein degradation (ERAD) and autophagy] to ensure only correctly folded proteins exit the ER and enter the cis-Golgi compartment. Perturbation of protein folding in the ER activates the unfolded protein response (UPR) that evolved to increase ER protein folding capacity and efficiency and degrade misfolded proteins. Accumulation of misfolded proteins in the ER to a level that exceeds the ER-chaperone folding capacity is a major factor that exacerbates protein aggregation. The most significant ER resident protein that prevents protein aggregation in the ER is the heat shock protein 70 (HSP70) homologue, BiP/GRP78, which is a peptide-dependent ATPase that binds unfolded/misfolded proteins and releases them upon ATP binding. Since exogenous factors can also reduce protein misfolding and aggregation in the ER, such as chemical chaperones and antioxidants, these treatments have potential therapeutic benefit for ER protein aggregation-associated diseases.

ER-associated degradation in health and disease - from substrate to organism.

The recent literature has revolutionized our view on the vital importance of endoplasmic reticulum (ER)-associated degradation (ERAD) in health and disease. Suppressor/enhancer of Lin-12-like (Sel1L)-HMG-coA reductase degradation protein 1 (Hrd1)-mediated ERAD has emerged as a crucial determinant of normal physiology and as a sentinel against disease pathogenesis in the body, in a largely substrate- and cell type-specific manner. In this Review, we highlight three features of ERAD, constitutive versus inducible ERAD, quality versus quantity control of ERAD and ERAD-mediated regulation of nuclear gene transcription, through which ERAD exerts a profound impact on a number of physiological processes.

Inhibition of ERAD synergizes with FTS to eradicate pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is driven by oncogenic KRAS mutations. Farnesyl thiosalicylic acid (FTS), also known as salirasib, is a RAS inhibitor that selectively dislodges active RAS proteins from cell membrane, inhibiting downstream signaling. FTS has demonstrated limited therapeutic efficacy in PDAC patients despite being well tolerated. METHODS: To improve the efficacy of FTS in PDAC, we performed a genome-wide CRISPR synthetic lethality screen to identify genetic targets that synergize with FTS treatment. Among the top candidates, multiple genes in the endoplasmic reticulum-associated protein degradation (ERAD) pathway were identified. The role of ERAD inhibition in enhancing the therapeutic efficacy of FTS was further investigated in pancreatic cancer cells using pharmaceutical and genetic approaches. RESULTS: In murine and human PDAC cells, FTS induced unfolded protein response (UPR), which was further augmented upon treatment with a chemical inhibitor of ERAD, Eeyarestatin I (EerI). Combined treatment with FTS and EerI significantly upregulated the expression of UPR marker genes and induced apoptosis in pancreatic cancer cells. Furthermore, CRISPR-based genetic ablation of the key ERAD components, HRD1 and SEL1L, sensitized PDAC cells to FTS treatment. CONCLUSION: Our study reveals a critical role for ERAD in therapeutic response of FTS and points to the modulation of UPR as a novel approach to improve the efficacy of FTS in PDAC treatment.

The inducible ß5i proteasome subunit contributes to proinsulin degradation in GRP94-deficient ß-cells and is overexpressed in type 2 diabetes pancreatic islets.

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when ß-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible ß5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of ß5i-containing intermediate proteasomes was significantly increased in these cells, as was ß5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon ß5i small interfering RNA-mediated knockdown. Finally, the fraction of ß-cells expressing the ß5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that ß5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.

Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches.

Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, we developed a Drosophila model of NGLY1 deficiency. Loss of NGLY1 function resulted in developmental delay and lethality. We used RNAseq to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. We show that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches.

S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein.

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins.

The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER to the proteasome. Still, however, little is known about the components mediating ERAD of membrane proteins. Here we show that the evolutionary conserved rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA+-ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by a shared mechanism.