ER-associated Degradation Pathway Research Articles

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.

The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.

Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome.

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.

The integration of single-cell and bulk RNA-seq atlas reveals ERS-mediated acinar cell damage in acute pancreatitis

BackgroundAcute pancreatitis (AP) is a clinically common acute abdominal disease, whose pathogenesis remains unclear. The severe patients usually have multiple complications and lack specific drugs, leading to a high mortality and poor outcome. Acinar cells are recognized as the initial site of AP. However, there are no precise single-cell transcriptomic profiles to decipher the landscape of acinar cells during AP, which are the missing pieces of jigsaw we aimed to complete in this study.MethodsA single-cell sequencing dataset was used to identify the cell types in pancreas of AP mice and to depict the transcriptomic maps in acinar cells. The pathways’ activities were evaluated by gene sets enrichment analysis (GSEA) and single-cell gene sets variation analysis (GSVA). Pseudotime analysis was performed to describe the development trajectories of acinar cells. We also constructed the protein–protein interaction (PPI) network and identified the hub genes. Another independent single-cell sequencing dataset of pancreas samples from AP mice and a bulk RNA sequencing dataset of peripheral blood samples from AP patients were also analyzed.ResultsIn this study, we identified genetic markers of each cell type in the pancreas of AP mice based on single-cell sequencing datasets and analyzed the transcription changes in acinar cells. We found that acinar cells featured acinar-ductal metaplasia (ADM), as well as increased endocytosis and vesicle transport activity during AP. Notably, the endoplasmic reticulum stress (ERS) and ER-associated degradation (ERAD) pathways activated by accumulation of unfolded/misfolded proteins in acinar cells could be pivotal for the development of AP.ConclusionWe deciphered the distinct roadmap of acinar cells in the early stage of AP at single-cell level. ERS and ERAD pathways are crucially important for acinar homeostasis and the pathogenesis of AP.

AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2.

Inactivating mutations of kidney Na-K-2Cl cotransporter NKCC2 lead to antenatal Bartter syndrome (BS) type 1, a life-threatening salt-losing tubulopathy. We previously reported that this serious inherited renal disease is linked to the endoplasmic reticulum-associated degradation (ERAD) pathway. The purpose of this work is to characterize further the ERAD machinery of NKCC2. Here, we report the identification of ancient ubiquitous protein 1 (AUP1) as a novel interactor of NKCC2 ER-resident form in renal cells. AUP1 is also an interactor of the ER lectin OS9, a key player in the ERAD of NKCC2. Similar to OS9, AUP1 co-expression decreased the amount of total NKCC2 protein by enhancing the ER retention and associated protein degradation of the cotransporter. Blocking the ERAD pathway with the proteasome inhibitor MG132 or the α-mannosidase inhibitor kifunensine fully abolished the AUP1 effect on NKCC2. Importantly, AUP1 knock-down or inhibition by overexpressing its dominant negative form strikingly decreased NKCC2 polyubiquitination and increased the protein level of the cotransporter. Interestingly, AUP1 co-expression produced a more profound impact on NKCC2 folding mutants. Moreover, AUP1 also interacted with the related kidney cotransporter NCC and downregulated its expression, strongly indicating that AUP1 is a common regulator of sodium-dependent chloride cotransporters. In conclusion, our data reveal the presence of an AUP1-mediated pathway enhancing the polyubiquitination and ERAD of NKCC2. The characterization and selective regulation of specific ERAD constituents of NKCC2 and its pathogenic mutants could open new avenues in the therapeutic strategies for type 1 BS treatment.

Casting Light on the Janus-Faced HMG-CoA Reductase Degradation Protein 1: A Comprehensive Review of Its Dualistic Impact on Apoptosis in Various Diseases.

Nowadays, it is well recognized that apoptosis, as a highly regulated cellular process, plays a crucial role in various biological processes, such as cell differentiation. Dysregulation of apoptosis is strongly implicated in the pathophysiology of numerous disorders, making it essential to comprehend its underlying mechanisms. One key factor that has garnered significant attention in the regulation of apoptotic pathways is HMG-CoA reductase degradation protein 1, also known as HRD1. HRD1 is an E3 ubiquitin ligase located in the endoplasmic reticulum (ER) membrane. Its primary role involves maintaining the quality control of ER proteins by facilitating the ER-associated degradation (ERAD) pathway. During ER stress, HRD1 aids in the elimination of misfolded proteins that accumulate within the ER. Therefore, HRD1 plays a pivotal role in the regulation of apoptotic pathways and maintenance of ER protein quality control. By targeting specific protein substrates and affecting apoptosis-related pathways, HRD1 could be an exclusive therapeutic target in different disorders. Dysregulation of HRD1-mediated processes contributes significantly to the pathophysiology of various diseases. The purpose of this review is to assess the effect of HRD1 on the pathways related to apoptosis in various diseases from a therapeutic perspective.

Direct binding to sterols accelerates endoplasmic reticulum-associated degradation of HMG CoA reductase

The maintenance of cholesterol homeostasis is crucial for normal function at both the cellular and organismal levels. Two integral membrane proteins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and Scap, are key targets of a complex feedback regulatory system that operates to ensure cholesterol homeostasis. HMGCR catalyzes the rate-limiting step in the transformation of the 2-carbon precursor acetate to 27-carbon cholesterol. Scap mediates proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a membrane-bound transcription factor that controls expression of genes involved in the synthesis and uptake of cholesterol. Sterol accumulation triggers binding of HMGCR to endoplasmic reticulum (ER)-localized Insig proteins, leading to the enzyme's ubiquitination and proteasome-mediated ER-associated degradation (ERAD). Sterols also induce binding of Insigs to Scap, which leads to sequestration of Scap and its bound SREBP-2 in the ER, thereby preventing proteolytic activation of SREBP-2 in the Golgi. The oxygenated cholesterol derivative 25-hydroxycholesterol (25HC) and the methylated cholesterol synthesis intermediate 24,25-dihydrolanosterol (DHL) differentially modulate HMGCR and Scap. While both sterols promote binding of HMGCR to Insigs for ubiquitination and subsequent ERAD, only 25HC inhibits the Scap-mediated proteolytic activation of SREBP-2. We showed previously that 1,1-bisphosphonate esters mimic DHL, accelerating ERAD of HMGCR while sparing SREBP-2 activation. Building on these results, our current studies reveal specific, Insig-independent photoaffinity labeling of HMGCR by photoactivatable derivatives of the 1,1-bisphosphonate ester SRP-3042 and 25HC. These findings disclose a direct sterol binding mechanism as the trigger that initiates the HMGCR ERAD pathway, providing valuable insights into the intricate mechanisms that govern cholesterol homeostasis.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Discovery of Natural Potent HMG-CoA Reductase Degraders for Lowering Cholesterol.

Exploitation of key protected wild plant resources makes great sense, but their limited populations become the major barrier. A particular strategy for breaking this barrier was inspired by the exploration of a resource-saving fungal endophyte Penicillium sp. DG23, which inhabits the key protected wild plant Schisandra macrocarpa. Chemical studies on the cultures of this strain afforded eight novel indole diterpenoids, schipenindolenes A-H (1-8), belonging to six diverse skeleton types. Importantly, semisyntheses suggested some key nonenzymatic reactions constructing these molecules and provided targeted compounds, in particular schipenindolene A (Spid A, 1) with low natural abundance. Remarkably, Spid A was the most potent HMG-CoA reductase (HMGCR) degrader among the indole diterpenoid family. It degraded statin-induced accumulation of HMGCR protein, decreased cholesterol levels and acted synergistically with statin to further lower cholesterol. Mechanistically, transcriptomic and proteomic profiling suggested that Spid A potentially activated the endoplasmic reticulum-associated degradation (ERAD) pathway to enhance the degradation of HMGCR, while simultaneously inhibiting the statin-activated expression of many key enzymes in the cholesterol and fatty acid synthesis pathways, thereby strengthening the efficacy of statins and potentially reducing the side effects of statins. Collectively, this study suggests the potential of Spid A for treating cardiovascular disease.

UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR.

The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.

Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function

Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using invivo and invitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.

ER-associated degradation in cystinosis pathogenesis and the prospects of precision medicine.

Cystinosis is a lysosomal storage disease that is characterized by the accumulation of dipeptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7Δ). We demonstrated that this mutant is retained in the ER and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the roles of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7Δ) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones previously used for treating CFTR F508Δ and demonstrated that they could facilitate the folding and trafficking of cystinosin(7Δ). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by approximately 70%. We believe that our study conclusively establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7Δ).

Boningmycin induces AMPK-mediated endoplasmic reticulum-associated degradation of PD-L1 protein in human cancer cells

Anti-PD-1/PD-L1 monoclonal antibodies have displayed remarkable clinical benefits and revolutionized the treatment of multiple tumor types, but the low response rates and immune-related adverse events limit their application, which promoting the development of small molecule agents to improve the efficacy of PD-1/PD-L1 blockade therapy. Boningmycin (BON), a new small molecule belonging to bleomycin (BLM) family, exhibits potent anticancer activity in vitro and in vivo, as well as negligible lung toxicity, thereby can be an alternative of BLM. However, understandings about the anticancer mechanism of BLM-related compounds are extremely rare, it remains unclear if they affect PD-L1 level in a manner similar to that of other antitumor drugs. In this study, we discover that BON significantly reduces PD-L1 protein level in NCI-H460 and HT-1080 cells. Meanwhile, BON decreases the protein level of PD-L1 in a tumor xenograft model of NCI-H460 cells. Nevertheless, the mRNA level is not influenced after BON exposure. Furthermore, BON-induced PD-L1 reduction is proteasome- dependent. By using specific inhibitors and RNA interference technology, we confirm that the decline of PD-L1 protein by BON is mediated by AMPK-activated endoplasmic reticulum-associated degradation pathway, which is like to the action of metformin. Last but not the least, BON has synergism on gefitinib in vitro and in vivo. In conclusion, it is the first report demonstrating that BON decreases PD-L1 protein level through AMPK-mediated endoplasmic reticulum-associated degradation pathway. These findings will benefit the clinical transformation of BON and aid in the elucidation of molecular mechanism of BLM-related compounds.

Importance of N-Glycosylation for the Expression and Function of Human Organic Anion Transporting Polypeptide 2B1.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a membrane transporter widely expressed in organs crucial for drug absorption and disposition such as the intestine, liver, and kidney. Evidence indicates that OATP2B1 is a glycoprotein. However, the sites of glycosylation and their contribution to the function and expression of OATP2B1 are largely unknown. In this study, by site-directed mutagenesis, we determined that two of four potential N-glycosylation sites in OATP2B1, N176 and N538, are indeed glycosylated. Functional studies revealed that the transport activities of mutants N176Q and N538Q were greatly reduced as compared to that of wild-type OATP2B1. However, the reduced activity was not due to the impairment of transport function per se but due to the decreased surface expression as the Km and normalized Vmax values of N176Q and N538Q were comparable to those of OATP2B1. Quantitative polymerase chain reaction (PCR) revealed that N176Q and N538Q mutations did not affect the expression of OATP2B1 at a transcriptional level. Immunofluorescence analysis showed that deglycosylated OATP2B1 was largely retained in the endoplasmic reticulum, which may activate the endoplasmic reticulum-associated degradation pathway, and the ubiquitin-proteasome system played a major role in the degradation of OATP2B1. Taken together, OATP2B1 is N-glycosylated, and N-glycosylation is essential for the surface expression of OATP2B1 but not critical for the transport function of OATP2B1 per se.

Identification of endoplasmic reticulum stress response genes in homologous vs. heterologous asf infections in vitro

Abstract The endoplasmic reticulum (ER) is crucial for the production, processing and transport of proteins. Infection with pathogens activates Unfolded Protein Response (UPR), which can lead to their survival/replication or elimination from the body. Although little is known about the role of the ER stress response in the pathogenesis of viral infections, the regulation of ER stress may be important in intractable infectious diseases. We conducted a comparative analysis of the expression of genes involved in ER stress response in peripheral blood mononuclear cells (PBMCs) from animals immunized with an attenuated strain of ASFV strain Congo-a (KK262) and then stimulated in vitro by two serologically different virulent strains Congo-v (K49) or Mozambique-v (M78), to expand our understanding of the early determinants of response to homologous and heterologous infection. We found up-regulation of genes of all three sensory molecules (PERK, ATF6 and IRE1) of UPR pathway in cells infected with only a homologous strain. For the first time, a number of up-regulated genes of the ER-associated degradation pathway (ERAD), which destroys misfolded proteins, were also detected. By understanding how viruses modify elements of cellular response to stress, we learn more about the pathogenesis, as well as how we can use it to prevent viral diseases.

Role of NFE2L1 in the Regulation of Proteostasis: Implications for Aging and Neurodegenerative Diseases.

A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis ("proteostasis") that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced "bounce-back effect" regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.

Orosomucoid proteins limit endoplasmic reticulum stress in plants.

Sphingolipids are cell membrane components and signaling molecules that induce endoplasmic reticulum (ER) stress responses, but the underlying mechanism is unknown. Orosomucoid proteins (ORMs) negatively regulate serine palmitoyltransferase activity, thus helping maintain proper sphingolipid levels in humans, yeast, and plants. In this report, we explored the roles of ORMs in regulating ER stress in Arabidopsis thaliana. Loss of ORM1 and ORM2 function caused constitutive activation of the unfolded protein response (UPR), as did treatment with the ceramide synthase inhibitor Fumonisin B1 (FB1) or ceramides. FB1 treatment induced the transcription factor bZIP28 to relocate from the ER membrane to the nucleus. The transcription factor WRKY75 positively regulates the UPR and physically interacted with bZIP28. We also found that the orm mutants showed impaired ER-associated degradation (ERAD), blocking the degradation of misfolded MILDEW RESISTANCE LOCUS-O 12 (MLO-12). ORM1 and ORM2 bind to EMS-MUTAGENIZED BRI1 SUPPRESSOR 7 (EBS7), a plant-specific component of the Arabidopsis ERAD complex, and regulate its stability. These data strongly suggest that ORMs in the ER membrane play vital roles in the UPR and ERAD pathways to prevent ER stress in Arabidopsis. Our results reveal that ORMs coordinate sphingolipid homeostasis with ER quality control and play a role in stress responses.

Transcriptome Sequencing Reveals the Response to Acute Thermal Stress in the Pacific Abalone, Haliotis discus hannai

The Pacific abalone is an economically important cold-water shellfish. With its widespread culture, high temperature has become a key abiotic factor for the high mortality of Pacific abalone in summer, particularly in the south of China. To understand the molecular regulatory mechanisms of thermal stress response in Pacific abalone for further analyzing its heat adaptive capacity, we subjected Pacific abalone to acute heat stress at 28°C for 6, 24, and 48 hr, respectively. A total of 2,213, 2,337, and 1,420 differentially expressed genes (DEGs) were identified in Pacific abalone after 6, 24, and 48 hr of thermal stress, respectively, as compared with the control group without heat stimulation. These DEGs were significantly enriched in protein folding and chaperone-mediated protein folding process, antigen processing and presentation, and protein processing in endoplasmic reticulum pathways. The expression of numerous molecular chaperones was significantly upregulated after thermal stress. Maintaining cellular homeostasis through the upregulated expression of molecular chaperones associated with endoplasmic reticulum-associated degradation (ERAD) pathways might be central to the defense of Pacific abalone against thermal stress. In addition, the expression of Toll-like receptor 6 (TLR6) and myeloid differentiation factor 88 (MyD88) genes of the TLR signaling pathway was also induced. Therefore, the Pacific abalone might activate the immune defense system to resist the invasion of pathogenic bacteria by upregulating immune-related genes after thermal challenges. Our results not only contribute to a deeper understanding of heat stress regulation in the Pacific abalone but also provide basic data for molecular genetic breeding of heat-resistant abalone.

The GRP78-PERK axis contributes to memory and synaptic impairments in Huntington's disease R6/1 mice

Increasing evidence indicates that a key factor in neurodegenerative diseases is the activation of the unfolded protein response (UPR) caused by an accumulation of misfolded proteins in the endoplasmic reticulum (ER stress). Particularly, in Huntington's disease (HD) mutant huntingtin (mHtt) toxicity involves disruption of the ER-associated degradation pathway and loss of the ER protein homeostasis leading to neuronal dysfunction and degeneration. Besides the role of the UPR in regulating cell survival and death, studies that demonstrate the contribution of sustained UPR activation, particularly of PERK signaling, in memory disturbances and synaptic plasticity deficiencies are emerging. Given the contribution of hippocampal dysfunction to emotional and cognitive deficits seen in HD, we have analyzed the involvement of ER stress in HD memory alterations. We have demonstrated that at early disease stages, ER stress activation manifested as an increase in GRP78 and CHOP is observed in the hippocampus of R6/1 mice. Genetic reduction of GRP78 expression resulted in preventing hippocampal-dependent memory alterations but no motor deficits. Accordingly, hippocampal neuropathology namely, dendritic spine loss and accumulation of mHtt aggregates was ameliorated by GRP78 reduction. To elucidate the signaling pathways, we found that the inactivation of PERK by GSK2606414 restored spatial and recognition memories in R6/1 mice and rescued dendritic spine density in CA1 pyramidal neurons and protein levels of some specific immediate early genes. Our study unveils the critical role of the GRP78/PERK axis in memory impairment in HD mice and suggests the modulation of PERK activation as a novel therapeutic target for HD intervention.