ER-associated Degradation Research Articles

Trafficking to the Cell Surface of Amino Acid Transporter SLC6A14 Upregulated in Cancer Is Controlled by Phosphorylation of SEC24C Protein by AKT Kinase.

Cancer cells need a constant supply of nutrients. SLC6A14, an amino acid transporter B0,+ (ATB0,+) that is upregulated in many cancers, transports all but acidic amino acids. In its exit from the endoplasmic reticulum (ER), it is recognized by the SEC24C subunit of coatomer II (COPII) for further vesicular trafficking to the plasma membrane. SEC24C has previously been shown to be phosphorylated by protein kinase B/AKT, which is hyper-activated in cancer; therefore, we analyzed the influence of AKT on SLC6A14 trafficking to the cell surface. Studies on overexpressed and endogenous transporters in the breast cancer cell line MCF-7 showed that AKT inhibition with MK-2206 correlated with a transient increase of the transporter in the plasma membrane, not resulting from the inhibition of ER-associated protein degradation. Two-dimensional electrophoresis demonstrated the decreased phosphorylation of SLC6A14 and SEC24C upon AKT inhibition. A proximity ligation assay confirmed this conclusion: AKT inhibition is correlated with decreased SLC6A14 phosphothreonine and SEC24C phosphoserine. Augmented levels of SLC6A14 in plasma membrane led to increased leucine transport. These results show that the inactivation of AKT can rescue amino acid delivery through SLC6A14 trafficking to the cell surface, supporting cancer cell survival. The regulation of the ER export of the amino acid transporter seems to be a novel function of AKT.

The novel ER stress inducer Sec C triggers apoptosis by sulfating ER cysteine residues and degrading YAP via ER stress in pancreatic cancer cells

Pancreatic adenocarcinoma (PAAD) is one of the most lethal malignancies. Although gemcitabine (GEM) is a standard treatment for PAAD, resistance limits its application and therapy. Secoemestrin C (Sec C) is a natural compound from the endophytic fungus Emericella, and its anticancer activity has not been investigated since it was isolated. Our research is the first to indicate that Sec C is a broad-spectrum anticancer agent and could exhibit potently similar anticancer activity both in GEM-resistant and GEM-sensitive PAAD cells. Interestingly, Sec C exerted a rapid growth-inhibiting effect (80% death at 6 h), which might be beneficial for patients who need rapid tumor shrinkage before surgery. Liquid chromatography/mass spectrometry and N-acetyl-l-cysteine (NAC) reverse assays show that Sec C sulfates cysteines to disrupt disulfide-bonds formation in endoplasmic reticulum (ER) proteins to cause protein misfolding, leading to ER stress and disorder of lipid biosynthesis. Microarray data and subsequent assays show that ER stress-mediated ER-associated degradation (ERAD) ubiquitinates and downregulates YAP to enhance ER stress via destruction complex (YAP–Axin–GSK–βTrCP), which also elucidates a unique degrading style for YAP. Potent anticancer activity in GEM-resistant cells and low toxicity make Sec C a promising anti-PAAD candidate.

Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β-selection.

Signals from the pre-T cell receptor and Notch coordinately instruct β-selection of CD4-CD8-double negative (DN) thymocytes to generate αβ T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by β-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during β-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse αβ T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1α/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency-induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.

ER-Phagy: A New Regulator of ER Homeostasis.

The endoplasmic reticulum (ER) is one of the most important cellular organelles and is essential for cell homeostasis. Upon external stimulation, ER stress induces the unfolded protein response (UPR) and ER-associated degradation (ERAD) to maintain ER homeostasis. However, persistent ER stress can lead to cell damage. ER-phagy is a selective form of autophagy that ensures the timely removal of damaged ER, thereby protecting cells from damage caused by excessive ER stress. As ER-phagy is a newly identified form of autophagy, many receptor-mediated ER-phagy pathways have been discovered in recent years. In this review, we summarize our understanding of the maintenance of ER homeostasis and describe the receptors identified to date. Finally, the relationships between ER-phagy and diseases are also discussed.

Roles of XBP1s in Transcriptional Regulation of Target Genes.

The spliced form of X-box binding protein 1 (XBP1s) is an active transcription factor that plays a vital role in the unfolded protein response (UPR). Under endoplasmic reticulum (ER) stress, unspliced Xbp1 mRNA is cleaved by the activated stress sensor IRE1α and converted to the mature form encoding spliced XBP1 (XBP1s). Translated XBP1s migrates to the nucleus and regulates the transcriptional programs of UPR target genes encoding ER molecular chaperones, folding enzymes, and ER-associated protein degradation (ERAD) components to decrease ER stress. Moreover, studies have shown that XBP1s regulates the transcription of diverse genes that are involved in lipid and glucose metabolism and immune responses. Therefore, XBP1s has been considered an important therapeutic target in studying various diseases, including cancer, diabetes, and autoimmune and inflammatory diseases. XBP1s is involved in several unique mechanisms to regulate the transcription of different target genes by interacting with other proteins to modulate their activity. Although recent studies discovered numerous target genes of XBP1s via genome-wide analyses, how XBP1s regulates their transcription remains unclear. This review discusses the roles of XBP1s in target genes transcriptional regulation. More in-depth knowledge of XBP1s target genes and transcriptional regulatory mechanisms in the future will help develop new therapeutic targets for each disease.

Gallic Acid Prevents the Oxidative and Endoplasmic Reticulum Stresses in the Hippocampus of Adult-Onset Hypothyroid Rats.

Thyroid hormone is essential for hippocampal redox environment and neuronal viability in adulthood, where its deficiency causes hypothyroidism related to oxidative and endoplasmic reticulum stresses in the hippocampus, resulting in neuronal death. One option of treatment is antioxidants; however, they must be transported across the blood-brain barrier. Gallic acid is a polyphenol that meets these criteria. Thus, this study aimed to prove that the neuroprotective mechanism of GA is associated with the prevention of oxidative and endoplasmic reticulum stresses in the hippocampus of adult-onset hypothyroid rats. Male Wistar rats were divided into euthyroid (n = 20) and hypothyroid groups (n = 20). Thyroidectomy with parathyroid gland reimplementation caused hypothyroidism. Each group was subdivided into two: vehicle and 50 mg/kg/d of gallic acid. 3 weeks after thyroidectomy, six animals of each group were euthanized, and the hippocampus was dissected to evaluate oxidative and endoplasmic reticulum stress markers. The rest of the animals were euthanized after 4 weeks of treatment for histological analysis of the hippocampus. The results showed that hypothyroidism increased lipid peroxidation, reactive oxygen species, and nitrites; it also increased endoplasmic reticulum stress by activating the inositol-requiring enzyme-1α (IRE1α) pathway, the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activated transcription factor 6α (ATF6α) pathways associated with a proapoptotic state that culminates in hippocampal neuronal damage. Meanwhile, the hypothyroid rat treated with gallic acid reduced oxidative stress and increased endoplasmic reticulum-associated degradation (ERAD) through IRE1α and ATF6. Also, the gallic acid treatment prevented the Bax/BCl2 ratio from increasing and the overexpression of p53 and caspase 12. This treatment in hypothyroid animals was associated with the neuronal protection observed in the hippocampus. In conclusion, gallic acid prevents hypothyroidism-induced hippocampal damage associated with oxidative and endoplasmic reticulum stresses.

Abstract 2155: BRCAness in HGSC cancer initiation

Abstract Introduction: The luteal phase of BRCA mutation carrier tissues has distinct profiles of differentially expressed genes compared to wild-type fimbria. High Grade Serous Ovarian Carcinoma (HGSOC) is one of the most lethal gynecological diseases and presents at late tumor stage. Studies focusing on the early events of the disease have implicated the fimbriated end of the fallopian tube as the high-risk zone for cancer development. In this study, we aim to identify BRCA transcriptomic signatures in the fimbria of patients with a BRCA mutation and compare the luteal and follicular phases to capture the effect of the extrinsic milieu in response to stressors. Experimental Procedures: Laser capture microdissection (LCM) was performed on 68 formalin fixed paraffin embedded (FFPE) fallopian tube samples based on BRCA mutation status and ovulatory cycle: Normal-Follicular, Normal-Luteal, BRCA-Follicular, BRCA-Luteal. Nine HGSC samples were derived from patients with a mutation in BRCA1. Six-Twelve, 10um FFPE sections were cut and stained with hematoxylin prior to LCM. RNA was extracted using the Roche High Pure FFPE Micro Kit and samples and were processed using Illumina Tru-Seq Stranded Total RNA Kit with RiboGold ready and sequenced on the Illumina Hi-seq 2000 V3. Bioinformatic analysis performed on multiple comparisons highlighted differentially expressed genes. Gene Set Enrichment Analysis (GSEA) performed on subgroups highlighted pathways of interest for each comparison. Genes highly expressed or strongly suppressed were shortlisted for further analysis. Western blot analysis performed on wildtype patients (WT), BRCA1 mutation and BRCA2 mutation showed protein expression of selected genes. Results: The fimbriae compared to ampullae have more stem-like features, have up-regulated antioxidant/xenobiotic genes, and have more inflammatory cells intercalated within the epithelia. The fimbria of FTE-BRCA have distinctly up-regulated genes involved in DDR (DNA damage response) which are exacerbated in the luteal phase of the ovulatory cycle. BRCA1/2 luteal versus follicular phases are involved in endoplasmic reticulum stress, migration, MAPKK. Whereas decreased differentially expressed genes are involved with G2/M checkpoint, DNA replication and ER-associated degradation pathway. Post-ovulatory phase of the ovarian cycle drives inflammation, differentiation and DNA damage-repair in FTE cells. Furthermore, GSEA analysis of the luteal and follicular phases showed positive enrichment of the inflammatory response pathway and negative enrichment of oxidative phosphorylation in the luteal phase. Conclusions: Our results highlight distinct pathways related to repair and inflammation for the BRCA1 mutation carriers and luteal phase of the fimbria. These results are consistent with previous examination of the fallopian tube and provide insight into targetable genes associated with the development of HGSC. Citation Format: Ramlogan Sowamber, Iru Paudel, Alex Sanchez, Alexandra Diaz, Patricia Shaw, Matthew Schlumbrecht, Sophia George. BRCAness in HGSC cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2155.

Hypercapnia Induces Inositol-Requiring Enzyme 1α-Driven Endoplasmic Reticulum-associated Degradation of the Na,K-ATPase β-Subunit.

Acute respiratory distress syndrome is often associated with elevated levels of CO2 (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA β-subunit (NKA-β) in the ER. Exposing murine precision-cut lung slices and human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid decrease of NKA-β abundance in the ER and at the cell surface. Knockdown of ER mannosidase α class 1B member 1 and ER degradation-enhancing α-mannosidase like protein 1 by siRNA or treatment with the mannosidase α class 1B member 1 inhibitor kifunensine rescued loss of NKA-β in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α), and treatment with an siRNA against IRE1α prevented the decrease of NKA-β in the ER. Of note, the hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1α in precision-cut lung slices and A549 cells, suggesting that Ca2+ efflux from the ER might be responsible for IRE1α activation and ERAD of NKA-β. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia.

Selection of CHO host and recombinant cell pools by inhibition of the proteasome results in enhanced product yields and cell specific productivity

Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER-associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc-fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins.

N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

Efficient degradation of by‐products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER‐associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER‐phagy receptors for ER‐to‐lysosome‐associated degradation (ERLAD). Demannosylation of N‐linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro‐collagen that cycles of de‐/re‐glucosylation of selected N‐glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD‐resistant misfolded proteins for FAM134B‐driven lysosomal delivery. In summary, we show that mannose and glucose processing of N‐glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.

Posttranslational Regulation of HMG CoA Reductase, the Rate-Limiting Enzyme in Synthesis of Cholesterol.

The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.

Differential protein abundance of vetiver grass in response to acid mine drainage.

Acid mine drainage (AMD) is an acidic and metalliferous discharge that imposes oxidative stress on living things through bioaccumulation and physical exposure. The abandoned Tab-Simco mining site of Southern Illinois generates highly acidic AMD with elevated sulfate (SO4 2- ) and various metals. Vetiver grass (Chrysopogon zizanioides) is effective for the remediation of Tab-Simco AMD at both mesocosm and microcosm levels over extended periods. In this study, we conducted a proteomic investigation of vetiver shoots under short and long-term exposure to AMD. Our objective was to decipher the physiological responses of vetiver to the combined abiotic stresses of AMD (metal and low pH). Differential regulation was observed for longer-term (56 days) exposure to AMD, which resulted in 17 upregulated and nine downregulated proteins, whereas shorter-term (7 days) exposure led to 14 upregulated and 14 downregulated proteins. There were significant changes to photosynthesis, including upregulation of electron transport chain proteins for light-dependent reactions after 56 days, whereas differential regulation of enzymes relating to C4 carbon fixation was observed after 7 days. Significant changes in amino acid and nitrogen metabolism, including upregulation of ethylene and flavonoid biosynthesis, along with plant response to nitrogen starvation, were observed. Short-term changes also included upregulation of glutathione reductase and methionine sulfoxide reductase, whereas longer-term changes included changes in protein misfolding and ER-associated protein degradation for stress management and acclimation.

ERAD components Derlin-1 and Derlin-2 are essential for postnatal brain development and motor function

SummaryDerlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function.

Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

Asaronic Acid Inhibits ER Stress and Ameliorates Impaired ERAD in 7Β-Hydroxycholesterol-Loaded Macrophages

Abstract Objectives Misfolded proteins were formed in the endoplasmic reticulum (ER) due to diverse stresses including metabolic stress and oxidative stress. Accumulation of unfolded proteins in the ER stimulates chaperone expression and ER-associated degradation (ERAD) process. This process involves the recognition of misfolded proteins to maintain the protein quality control, which in turn eliminates in association with the ER membrane. Upregulation of ubiquitination enzymes is an essential mechanism by which ER stress enhances ERAD. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. This study attempted to examine whether asaronic acid attenuated the 7Β-hydroxycholesterol-elicited ER stress of macrophages. Methods J774A.1 murine macrophage was incubated with 28 μM 7Β-hydroxycholesterol in absence and presence of 1–20 μΜ asaronic acid up to 24 h. Cytotoxicity was assessed by MTT assay. Expression levels of ER stress-responsive chaperones and ERAD biomarkers were measured by Western blot analysis and immunocytochemical staining with a specific antibody. Results Asaronic acid at 1–20 μM had a cytoprotective effect on macrophages against 7Β-hydroxycholesterol-induced toxicity. Asaronic acid diminished the induction and activation of ER stress sensors such as Grp/BiP, IRE1, and PERK in macrophages exposed to 7Β-hydroxycholesterol. Also, asaronic acid positively influenced the induction of ERAD process-linked components of EDEM1, OS9, SEl1L, HRD1, and VCP1/p97. Furthermore, asaronic acid promoted subsequent degradation reduced by 7Β-hydroxycholesterol via the cytosolar ubiquitin-proteasome system of macrophages. Conclusions These results demonstrate that asaronic acid attenuated 7Β-hydroxycholesterol-induced ER stress and improved impaired ER stress-mediated degradation systems. Therefore, asaronic acid may be a potent agent protecting macrophages against pathological ER stress damage. Funding Sources This work was supported by the BK21 FOUR(Fostering Outstanding Universities for Research, 4220200913807) funded by the National Research Foundation of Korea (NRF).

Integrated signaling system under endoplasmic reticulum stress in eukaryotic microorganisms.

The endoplasmic reticulum (ER) is a multifunctional organelle, which is crucial for correct folding and assembly of secretory and transmembrane proteins. Perturbations of ER function can cause ER stress. ER stress can activate the unfolded protein response (UPR) to cope with the accumulation of misfolded proteins and protein toxicity. UPR is a coordination system that regulates transcription and translation, leading to the recovery of ER homeostasis or cell death. However, cells have an integrated signaling system to cope with ER stress, which helps cells to restore and balance their ER function. The main components of this system are ER-associated degradation (ERAD), autophagy, hypoxia signaling, and mitochondrial biogenesis. If the balance cannot be restored, the imbalance will lead to cell death or apoptosis, or even to a series of diseases. In this review, a series of activities to restore the homeostasis of cells during ER stress are discussed. KEY POINTS: • Endoplasmic reticulum (ER) plays a key role in the biological process of cells. • Perturbations of ER function can cause ER stress, including the ER overload response (EOR), sterol-regulated cascade reaction, and the UPR. • Cells have an integrated signaling system (ERAD, autophagy, hypoxia signaling, and mitochondrial biogenesis) to cope with the adverse impact caused by ER stress.

A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7.

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.

The Impact of Endoplasmic Reticulum-Associated Protein Modifications, Folding and Degradation on Lung Structure and Function.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and induces the unfolded protein response (UPR) and other mechanisms to restore ER homeostasis, including translational shutdown, increased targeting of mRNAs for degradation by the IRE1-dependent decay pathway, selective translation of proteins that contribute to the protein folding capacity of the ER, and activation of the ER-associated degradation machinery. When ER stress is excessive or prolonged and these mechanisms fail to restore proteostasis, the UPR triggers the cell to undergo apoptosis. This review also examines the overlooked role of post-translational modifications and their roles in protein processing and effects on ER stress and the UPR. Finally, these effects are examined in the context of lung structure, function, and disease.

Regulation of Wnt/PCP signaling through p97/VCP-KBTBD7-mediated Vangl ubiquitination and endoplasmic reticulum-associated degradation.

The four-pass transmembrane proteins Vangl1 and Vangl2 are dedicated core components of Wnt/planar cell polarity (Wnt/PCP) signaling that critically regulate polarized cell behaviors in many morphological and physiological processes. Here, we found that the abundance of Vangl proteins is tightly controlled by the ubiquitin-proteasome system through endoplasmic reticulum-associated degradation (ERAD). The key ERAD component p97/VCP directly binds to Vangl at a highly conserved VCP-interacting motif and recruits the E3 ligase KBTBD7 via its UBA-UBX adaptors to promote Vangl ubiquitination and ERAD. We found that Wnt5a/CK1 prevents Vangl ubiquitination and ERAD by inducing Vangl phosphorylation, which facilitates Vangl export from the ER to the plasma membrane. We also provide in vivo evidence that KBTBD7 regulates convergent extension during zebrafish gastrulation and functions as a tumor suppressor in breast cancer by promoting Vangl degradation. Our findings reveal a previously unknown regulatory mechanism of Wnt/PCP signaling through the p97/VCP-KBTBD7-mediated ERAD pathway.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.