Multiple myeloma (MM) is an incurable plasma cell neoplasm that is highly reliant on endoplasmic reticulum-associated degradation (ERAD) to maintain protein homeostasis. Disrupting ERAD has been proposed as a therapeutic strategy to overcome proteasome inhibitor resistance; however, the identification of novel inhibitors has been limited. To address this, we conducted a cell-based high-throughput screen using the FDA repurposing library and identified omaveloxolone (RTA408) as a potent ERAD inhibitor that selectively impairs the degradation of ER luminal and membrane substrates, without affecting the degradation of key cytosolic proteins that are implicated in disease relapse. Surprisingly, although ER stress response pathways are activated after ERAD inhibition in MM, we find that apoptosis is mediated by altered lipid raft organization, leading to aberrant activation of the death-inducing signaling complex (DISC) and caspase 8 in the extrinsic apoptotic pathway. Notably, ERAD inhibition by RTA408 is cytotoxic to primary malignant plasma cells, including those resistant to proteasome inhibitors, and demonstrates in vivo anti-myeloma activity. Our findings establish a novel ERAD inhibitor, which is a valuable tool to dissect ERAD biology, and provide pre-clinical evidence for RTA408 as a therapeutic agent in MM.

The endoplasmic reticulum membrane protein complex (EMC) is an evolutionarily conserved multi-subunit complex. Due to its essential roles in protein biogenesis and quality control, the EMC has attracted considerable attention in recent years. In this review, we systematically explore the functions and disease-associated regulatory mechanisms of the EMC across various organ systems. We highlight the lung as a paradigmatic model for illustrating the 'molecular switch' function of EMC shaped by spatiotemporal and cell-type-specific contexts. Dysfunction of EMC contributes to pathologies and cancers of diverse organs, positioning EMC subunits as potential biomarkers and therapeutic targets. Despite considerable progress, our understanding of the molecular underpinnings of EMC in health and disease remains far from complete. Future efforts should aim to unravel the regulatory networks centered on EMC to harness their potential for cross-disease therapy development.

VLDLs are crucial for maintaining liver and whole-body lipid homeostasis. Limited knowledge exists regarding the lipidation process of VLDL. Endoplasmic reticulum (ER) luminal lipid droplets (LLDs) have been suggested to provide lipids for VLDL lipidation and maturation. Seipin, an integral membrane protein of the ER, plays key roles in the formation of cytoplasmic LDs (CLDs) and adipocyte differentiation. Surprisingly, seipin is hardly detectable in hepatocytes. Given the critical contribution of seipin in forming CLDs, we hypothesize that the absence of seipin in hepatocytes might ensure the proper formation of LLDs and the lipidation and assembly of VLDLs. To explore the functional interactions between CLDs, LLDs, and VLDLs, we generated liver-specific human seipin (hSeipin) overexpression (adeno-associated virus [AAV]-hSeipin) mice using AAV. We examined hepatic lipid accumulation, plasma lipid levels, VLDL lipidation, and liver pathology using biochemical, histological, and electron microscopy techniques. Liver-specific overexpression of seipin resulted in increased accumulation of CLDs in hepatocytes, accompanied by reduced plasma triacylglycerol and cholesterol levels. VLDL lipidation was severely impaired in AAV-hSeipin mice. When subjected to a high-fat, high-cholesterol diet, AAV-hSeipin mice developed more severe hepatic inflammation and fibrosis. These findings suggest that enhanced formation of CLDs driven by seipin may channel lipid storage toward the cytoplasm of hepatocytes, thereby impeding the biogenesis of LLDs and causing defective VLDL lipidation in the ER lumen. Our results thus provide important new insights into the connection between the biogenesis of CLDs and LLDs as well as VLDL assembly.

RCN2 facilitates esophageal squamous cellular carcinoma metastasis and cisplatin resistance through UBR5-mediated PPP2CA ubiquitination and degradation.

The two-pore domain K+ channel TWIK1-related alkalinisation-activated K+ channel 2 (TALK-2) is encoded by KCNK17, which is one of the most abundant beta cell K+ channel transcripts that also shows high islet expression specificity. Polymorphisms that increase islet KNCK17 expression or result in TALK-2 gain-of-function are associated with a predisposition for developing type 2 diabetes. However, there is a gap in knowledge of the beta cell function(s) of TALK-2. As K+ channels typically control beta cell Ca2+ handling, we aimed to examine the TALK-2 channel control of beta cell Ca2+ homeostasis and the resulting impact on insulin secretion. Localisation of TALK-2 was evaluated with immunofluorescent staining as well as TALK-2-GFP construct co-expressed with intracellular markers. TALK-2 function was evaluated by measuring changes in cytoplasmic Ca2+ (Ca2+C), endoplasmic reticulum Ca2+ (Ca2+ER), ER membrane potential (Vm), K+ currents and insulin secretion in a TALK-2 inducible cell line and/or primary human beta cells with adenoviral-mediated shRNA knockdown (KD) of TALK-2 or scramble shRNA. TALK-2 protein localised to the plasma membrane and ER membrane, and formed functional channels on the ER membrane. Ca2+ER release was accelerated by TALK-2 (slope for TALK-2-expressing cells vs controls: 14.8±0.7 vs 8.9±1.3, respectively, shown as mean±SE), which reduced Ca2+ER storage (ΔCa2+ER amplitude: TALK-2-expressing cells reduced by 25±5%) and increased basal relative Ca2+C (fold increase by 12±2%). Furthermore, TALK-2 diminished ER membrane hyperpolarisation following Ca2+ER release (Accelerated Sensor of Action Potentials [ASAP3ER] amplitude decreased by 20±0.8% in TALK-2-expressing cells), suggesting that TALK-2 strengthens the electrical driving force for Ca2+ER leak. In human beta cells, TALK-2-KD increased Ca2+ER stores by reducing Ca2+ER leak (2.30±0.12 vs controls 2.65±0.14). Moreover, TALK-2-KD reduced beta cell Ca2+C at euglycaemic conditions (2.88±0.36 vs controls 3.16±0.36) and increased beta cell Ca2+C influx in response to hyperglycaemic conditions (4.07±0.55 vs controls 3.45±0.48). Human pseudoislets with beta cell-specific TALK-2-KD displayed reduced basal insulin secretion (0.266±0.065 vs controls 0.432±0.073) and enhanced glucose-stimulated insulin secretion (GSIS; 85.01±13.96 vs controls 42.53±5.52). These data support the notion that TALK-2 functions on the human beta cell ER membrane to increase the electrical driving force for beta cell Ca2+ER release, reduces glucose-stimulated Ca2+ influx and limits GSIS. Furthermore, TALK-2-mediated amplification of Ca2+ER leak likely enhances basal insulin secretion by increasing Ca2+C. Therefore, polymorphisms in KCNK17 that increase TALK-2 activity or expression would be predicted to increase type 2 diabetes risk by blunting beta cell glucose-stimulated Ca2+ influx, limiting GSIS, promoting Ca2+ER leak and elevating basal insulin secretion.

Orchestration of lipid production, storage and mobilization is vital for cellular and systemic homeostasis1,2. Dysfunctional plasma lipid control represents the major risk factor for cardiometabolic diseases-the leading cause of human mortality3,4. Within the cellular landscape, the endoplasmic reticulum (ER) is the central hub of lipid synthesis and secretion, particularly in metabolically active hepatocytes in the liver or enterocytes in the gut5,6. Initially assembled in the ER lumen, lipid-ferrying lipoproteins necessitate the cross-membrane transfer of both neutral and phospholipids onto the lumenal apolipoprotein B (APOB), in a poorly defined process7-10. Here we show that the ER protein CLCC1 regulates cellular lipid partition and, consequently, systemic lipid homeostasis by participating in trans-bilayer equilibration of phospholipids. CLCC1 partners with the phospholipid scramblase TMEM41B11,12 to recognize imbalanced bilayers and promote lipid scrambling, thereby supporting lipoprotein biogenesis and the subsequent bulk lipid transport. Loss of CLCC1 or TMEM41B leads to the emergence of giant lumenal lipid droplets enclosed by imbalanced ER bilayers and, consequently, accelerated pathogenesis of metabolic-dysfunction-associated liver steatohepatitis. The results reveal that phospholipid scrambling at the ER is essential for establishing a dynamic equilibrium. Considering the requirement of trans-bilayer phospholipid equilibration in numerous biological processes, ranging from catabolic autophagy to viral infection13-16, we anticipate that future work will elucidate a homeostatic control mechanism intrinsic to ER function in lipid biogenesis and distribution.

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

Endoplasmic reticulum-mediated protein quality control (ERQC) safeguards secretory pathway proteostasis by recognizing, retaining, repairing, and removing misfolded proteins, and is therefore essential for plant growth, development, and stress tolerance. This system relies on ER-associated degradation (ERAD), in which irreparably misfolded proteins are first recognized in the ER, then exported across the ER membrane to the cytosol, where they are ubiquitinated by ER membrane-anchored ubiquitin ligases, and subsequently degraded by the cytosolic proteasome. Studies in yeast and mammals have defined several conserved ERAD branches, including a multiprotein ERAD complex centered on the polytopic ER membrane E3 ligase HMG-CoA reductase degradation protein 1 (Hrd1), which integrates substrate recognition, membrane retrotranslocation, ubiquitin conjugation, and cytosolic extraction. Recent advances in Arabidopsis show that plants retain the core Hrd1 ERAD architecture while incorporating additional regulatory elements that adapt this machinery to plant-specific physiological demands. Genetic and biochemical analyses of misfolded receptor kinases and engineered substrates have uncovered conserved and plant-specific components of the plant Hrd1 complex, revealing how the plant ERAD pathway integrates ERQC with hormone signaling, stress adaptation, immune responses, and growth regulation. This review synthesizes recent advances in plant ERAD research and highlights key conceptual and mechanistic questions that remain to be resolved.

Many physicochemical properties in the cellular milieu are important for cell function and survival. However, the polarity of different subcellular compartments and its role in protein condensate and aggregate formation within cells are less characterized. Here, we develop a method to compare the polarity in different subcellular compartments using the same polarity-sensitive solvatochromic fluorescent probe. Unexpectedly, the endoplasmic reticulum (ER) lumen displays a higher polarity and a more crowded environment than the cytosol in human cells. Polarity-decreasing and crowding-increasing hypertonic conditions induce condensate or aggregate formation of two intrinsically disordered proteins, with-no-lysine kinase 1 and Huntingtin gene (Htt) exon1 with an expanded polyQ stretch (Htt-polyQ), in the cytosol. However, targeting Htt-polyQ to the ER prevents its aggregation, suggesting that polarity but not crowding is more relevant to protein aggregation. Our results reveal the heterogeneity in subcellular polarity and crowding, and uncover previously unrecognized high-polarity in the ER lumen, which provides a unique environment for maintaining robust proteostasis.

Durable rust resistance in wheat conferred by engineering host protein TaHRLI to evade recognition by the virulence effector PstCRT.

The SEL1L-HRD1 complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD), a critical quality-control pathway that clears misfolded ER proteins. However, the molecular organization and pathogenic mechanisms of mammalian ERAD have remained elusive. Here, we report the cryo-EM structure of the core mammalian ERAD complex, comprising the ER lectin OS9, SEL1L, and the E3 ubiquitin ligase HRD1. The structure, validated by mutagenesis and crosslinking assays, reveals a dimeric assembly of the core complex in which SEL1L and OS9 form a claw-like configuration in the ER lumen that mediates substrate engagement, while HRD1 dimerizes within the membrane that may facilitate substrate translocation. Furthermore, pathogenic SEL1L mutations at the SEL1L-OS9 (Gly585Asp) and SEL1L-HRD1 (Ser658Pro) interfaces disrupt complex formation and impair ERAD activity. A newly identified disease-associated HRD1 variant (Ala91Asp), located in transmembrane helix 3, impairs HRD1 dimerization and substrate processing. These findings provide structural and functional insights for mammalian SEL1L-HRD1 ERAD and elucidate how mutations destabilizing this machinery contribute to human disease.

Abstract Background The endoplasmic reticulum (ER) is a dynamic and essential cell organelle involved in the synthesis and correct folding of secretory and membrane-bounded proteins, as well as in preserving intracellular Ca 2+ equilibrium. Main body During cellular stress, the deposition of unfolded or misfolded proteins in the ER lumen aggravates the cascade of the unfolded protein response (UPR), thus predisposing to ER stress. The interplay between ER and mitochondria exerts a pivotal role in coordinating intracellular Ca 2 ⁺ signaling, lipid transfer, mitochondrial dynamics, autophagy initiation, and apoptotic processes. Persistent or unresolved ER stress could predispose to cellular perturbations as well as numerous disease pathogeneses, such as neurodegenerative, renal, hepatic, reproductive, and neoplastic disorders. Increasing evidence suggests that natural compounds possess therapeutic potential by modulating ER stress pathways. For example, curcumin and resveratrol can alleviate ER stress by enhancing protein-folding capacity, reducing oxidative stress, and regulating UPR signaling. These bioactive molecules can either mitigate ER stress and restore proteostasis or, conversely, intensify ER stress and apoptosis. Conclusion This review explores the multifaceted effects of natural products on ER stress-related mechanisms and their implications for disease treatment and prevention. Graphical abstract

Viral protein biogenesis underpins every viral life cycle stage, and elucidating these processes could reveal fundamental principles of virus–host interaction, and vulnerabilities amenable to therapeutic targeting. Here we apply biophysical, molecular, and cell biology techniques to investigate the insertion, folding, and oligomerization of the SARS-CoV-2 M protein. We describe the sequential co-translational insertion of the hydrophobic core, and demonstrate that the cytosolic C-terminal domain undergoes slower adoption of its tertiary structure. Additionally, we characterize how the transmembrane domain bundle facilitates M-protein oligomerization. Our results reveal a hydrophobic residue cluster that is essential for protein folding and co-translational dimerization. Additionally, we identify the cellular machinery responsible for targeting and inserting the M protein into the ER membrane, and chaperones and cofactors that may contribute to proper folding.

Severe acute respiratory syndrome coronavirus (SARS-CoV) assembles and buds from the Golgi apparatus or the ER membrane, but the specific membrane microdomains utilized during this process remain underexplored. Here, we show that co-expression of the SARS-CoV structural proteins S, M, and N in HEK-293T cells is sufficient to generate genome-free SARS-CoV-like virus-like particles (VLPs), which preferentially bud from glycolipid-enriched membrane lipid raft microdomains. Immunofluorescence microscopy using raft-selective dyes (DiIC16) and spike-specific antibodies revealed strong co-localization of VLPs with lipid rafts. Detergent-resistant membrane analysis and sucrose gradient centrifugation further confirmed the presence of S protein in buoyant, raft-associated fractions alongside the raft marker CD44. Importantly, pharmacological disruption of rafts with methyl-β-cyclodextrin reduced VLP budding and S protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. Additionally, our data support lipid raft-associated proteins’ (e.g., FNRA, VIM, CD59, RHOA) roles in modulating cellular responses conducive to viral replication and assembly. These findings highlight lipid rafts as crucial platforms for SARS-CoV morphogenesis and suggest new avenues for vaccine and antiviral development using VLPs and raft-targeting therapeutics.

The activation of the NLRP3 inflammasome is a pivotal step in hyperinflammation in sepsis; however, the regulatory mechanisms underlying its activation are not fully understood. In this study, we found that 14-3-3ε facilitates NLRP3 inflammasome activation by enhancing NLRP3 K63 deubiquitination and promoting its translocation to the mitochondria-associated ER membranes (MAMs) for full activation. Mass spectrometry revealed that 14-3-3ε binds to NLRP3 in macrophages during sepsis. Plasma 14-3-3ε levels were elevated in patients with sepsis and were positively associated with disease severity. 14-3-3ε promoted NLRP3 inflammasome activation by facilitating NLRP3 aggregation and NLRP3-ASC assembly. The interaction between 14-3-3ε and NLRP3 was dependent on phosphorylation at the S194 site of NLRP3 NACHT domain. The NLRP3-14-3-3ε interaction promoted K63 deubiquitination and enhanced the translocation of NLRP3 to MAMs, which is necessary for full activation of NLRP3 inflammasome. Furthermore, macrophage-conditional KO of 14-3-3ε or treatment with BV02, a 14-3-3 inhibitor, improved the survival rate and alleviated organ injuries in septic mice. Taken together, our data indicate that 14-3-3ε functions as a positive regulator of the NLRP3 inflammasome and could be a target for sepsis treatment.

The endoplasmic reticulum (ER) orchestrates the folding of the large amounts of membrane and secretory proteins that are synthesized during the process of osteogenesis. The Unfolded Protein Response (UPR) resulting from accumulation of misfolded proteins in the ER lumen either promotes or inhibits osteoblast differentiation in vitro depending on magnitude and duration. All three transducers of the UPR, namely, IRE, PERK, and ATF6 proteins, have been implicated in skeletal biology, yet their specific contribution to osteoblast differentiation and function in vivo has not been investigated systematically. Here, the skeletal consequences of deleting each of them (i.e. Ire1α, Perk, or Atf6) in the osteoblast lineage using the Osx1-Cre transgene were determined. Mice with deletion of Ire1α in Osx1+ osteoblast precursors exhibited a marked reduction in osteoblast number, bone mass, and strength. Primary bone marrow cultures of osteoprogenitors lacking Ire1α had significantly reduced proliferation, alkaline phosphatase activity, and survival. Analyses of bulk RNA-seq data revealed suppression of osteogenic signature by Ire1α deletion in Osx1+ cells and predicted suppression of β-catenin activity. Mechanistically, Ire1α augments nuclear translocation and transcriptional activity of β-catenin in Osx1+ cells. In contrast, deletion of Perk or Atf6 genes in the osteoblast lineage using the Osx1-Cre transgene did not alter bone mass or strength. Collectively, these studies demonstrate that IRE1, but not other UPR transducers, promote physiological bone accrual in part by boosting β-catenin activity in osteoprogenitors.

The synthesis of the melanin pigment in melanocytes plays a crucial role in protecting the body from ultraviolet radiation. Tyrosinase, a key enzyme in melanogenesis, catalyzes the conversion of tyrosine to melanin in the melanosomes of melanocytes. During melanogenesis, Tyrosinase is abundantly synthesized in the lumen of the endoplasmic reticulum (ER) and subsequently transported from the ER to the melanosomes via the Golgi apparatus. In the present study, we demonstrate that Box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), an ER-resident transmembrane transcription factor that functions as an ER stress sensor, is activated by mild ER stress caused by abundant Tyrosinase synthesis. Activated BBF2H7 enhances COPII-mediated anterograde transport by inducing the expression of Sec23a, which is a COPII component and transcriptional target of BBF2H7. Loss of BBF2H7 attenuates the transport of Tyrosinase, leading to its accumulation in the ER lumen and reduced melanin production. Restoration of BBF2H7 or Sec23a expression in Bbf2h7-deficient melanocytes rescues anterograde transport of Tyrosinase from the ER and melanin pigmentation. Collectively, these findings reveal that the BBF2H7-Sec23a axis is essential for the ER-to-melanosome transport of Tyrosinase and subsequent melanin synthesis. Thus, it may be a prospective therapeutic target for disorders related to melanin pigmentation.

An endoplasmic reticulum-targeting NIR fluorescent probe for viscosity imaging in vitro and vivo.

In vitro methods for studying protein-lipid droplet interactions.

Selective autophagy of the endoplasmic reticulum (ER-phagy/reticulophagy) is essential for organelle homeostasis and host defense, yet how ER quality control (ERQC) pathways distinguish viral glycoproteins from misfolded host proteins remains poorly understood. Recent work identifies TMEM259/MEMBRALIN (transmembrane protein 259) as a selective ER-phagy receptor containing a non-canonical LC3-interacting region (LIR) motif that assembles a dedicated ER-to-lysosome-associated degradation (ERLAD) complex targeting viral class I fusion glycoproteins. TMEM259 is a multi-pass ER membrane protein with luminal domains that recruit MAN1B1 (mannosyl-oligosaccharide 1,2-α-mannosidase) and cytosolic regions that engage VCP/p97 (valosin-containing protein). This TMEM259-MAN1B1-VCP axis directs diverse viral glycoproteins, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, Ebola virus (EBOV) glycoprotein, influenza A virus (IAV) hemagglutinin (HA), and human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, to lysosomes in a ubiquitin-independent manner. In contrast, misfolded host glycoproteins are primarily cleared through canonical ER-associated degradation (ERAD) or alternative ERLAD pathways. Preferential recognition of densely glycosylated viral substrates suggests that MAN1B1 may function as a glycan-density sensor, enabling TMEM259 to couple ER proteostasis with intrinsic antiviral immunity. These findings expand the conceptual framework of selective autophagy and uncover a specialized ER-phagy pathway dedicated to eliminating viral glycoproteins.