Endoplasmic Reticulum Protein Quality Control Research Articles

Epi-Cyclophellitol Cyclosulfate, a Mechanism-Based Endoplasmic Reticulum α-Glucosidase II Inhibitor, Blocks Replication of SARS-CoV-2 and Other Coronaviruses.

The combined inhibition of endoplasmic reticulum (ER) α-glucosidases I and II has been shown to inhibit replication of a broad range of viruses that rely on ER protein quality control. We found, by screening a panel of deoxynojirimycin and cyclitol glycomimetics, that the mechanism-based ER α-glucosidase II inhibitor, 1,6-epi-cyclophellitol cyclosulfate, potently blocks SARS-CoV-2 replication in lung epithelial cells, halting intracellular generation of mature spike protein, reducing production of infectious progeny, and leading to reduced syncytium formation. Through activity-based protein profiling, we confirmed ER α-glucosidase II inhibition in primary airway epithelial cells, grown at the air-liquid interface. 1,6-epi-Cyclophellitol cyclosulfate inhibits early pandemic and more recent SARS-CoV-2 variants, as well as SARS-CoV and MERS-CoV. The reported antiviral activity is comparable to the best-in-class described glucosidase inhibitors, all competitive inhibitors also targeting ER α-glucosidase I and other glycoprocessing enzymes not involved in ER protein quality control. We propose selective blocking ER-resident α-glucosidase II in a covalent and irreversible manner as a new strategy in the search for effective antiviral agents targeting SARS-CoV-2 and other viruses that rely on ER protein quality control.

The dispensability of VH-VL pairing and the indispensability of VL domain integrity in the IgG1 secretion process.

Introduction: The CH1 domain of IgG antibodies controls assembly and secretion, mediated by the molecular chaperone BiP via the endoplasmic reticulum protein quality control (ERQC) mechanism. However, it is not clear whether the variable domains are necessary for this process. Methods: Here, we generated IgG1 antibodies in which the V domain (VH and/or VL) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells. Results: All Ig variants formed a covalent linkage between the Cγ1 and Cκ, were successfully secreted in an assembled form. Replacement of the cognate Vκ with a non-secretory pseudo Vκ (ψVκ) hindered secretion of individual or assembled secretion of neither heavy chains (HCs) nor light chains (LCs). The ψLC (ψVκ-Cκ) exhibited a less folded structure compared to the wild type (wt) LC, as evidenced by enhanced stable binding to the molecular chaperone BiP and susceptibility to proteolytic degradation. Molecular dynamics simulation demonstrated dramatic alterations in overall structure of ψFab (Fd-ψLC) from wt Fab. Discussion: These findings suggest that V domains do not initiate HC:LC assembly and secretion; instead, the critical factor governing IgG assembly and secretion is the CH-CL pairing. Additionally, the structural integrity of the VL domain is crucial for IgG secretion. These data offer valuable insight into the design of bioactive molecules based on an IgG backbone.

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.

Potential Role of Endoplasmic Reticulum Stress in Modulating Protein Homeostasis in Oligodendrocytes to Improve White Matter Injury in Preterm Infants.

Preterm white matter injury (WMI) is a demyelinating disease with high incidence and mortality in premature infants. Oligodendrocyte cells (OLs) are a specialized glial cell that produces myelin proteins and adheres to the axons providing energy and metabolic support which susceptible to endoplasmic reticulum protein quality control. Disruption of cellular protein homeostasis led to OLs dysfunction and cell death, immediately, the unfolded protein response (UPR) activated to attempt to restore the protein homeostasis via IRE1/XBP1s, PERK/eIF2α and ATF6 pathway that reduced protein translation, strengthen protein-folding capacity, and degraded unfolding/misfolded protein. Moreover, recent works have revealed the conspicuousness function of ER signaling pathways in regulating influenced factors such as calcium homeostasis, mitochondrial reactive oxygen generation, and autophagy activation to regulate protein hemostasis and improve the myelination function of OLs. Each of the regulation modes and their corresponding molecular mechanisms provides unique opportunities and distinct perspectives to obtain a deep understanding of different actions of ER stress in maintaining OLs' health and function. Therefore, our review focuses on summarizing the current understanding of ER stress on OLs' protein homeostasis micro-environment in myelination during white matter development, as well as the pathophysiology of WMI, and discussing the further potential experimental therapeutics targeting these factors that restore the function of the UPR in OLs myelination function.

Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes Ubc6 and Ubc7 confer resistance to hygromycin B in Saccharomyces cerevisiae.

Aberrant endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed through ER-associated degradation (ERAD) and INM-associated degradation (INMAD). We previously showed the Hrd1, Doa10, and Asi ERAD and INMAD ubiquitin ligases (E3s) in Saccharomyces cerevisiae confer resistance to hygromycin B, which distorts the ribosome decoding center. Here, we assessed the requirement of Ubc6 and Ubc7, the primary ERAD and INMAD ubiquitin-conjugating enzymes (E2s) for hygromycin B resistance. Loss of either E2 sensitized cells to hygromycin B, with UBC7 deletion having a greater impact, consistent with characterized roles for Ubc6 and Ubc7 in ER and INM protein quality control.

New mode of action of curcumin on prostate cancer cells: Modulation of endoplasmic reticulum-associated degradation mechanism and estrogenic signaling.

Prostate cancer is leading to cancer-related mortality in numerous men each year worldwide. While there are several treatment options, acquired drug resistance mostly limits the success of treatments. Therefore, there is a need for the development of innovative treatments. Curcumin is one of the bioactive polyphenolic ingredients identified in turmeric and has numerous biological activities, such as anti-inflammatory and anticancer. In the present study, we investigated the effect of curcumin on the ER-associated degradation (ERAD) and estrogenic signaling in prostate cancer cells. The antiproliferative effect of curcumin on human androgen-dependent prostate cancer cell lines LNCaP and VCaP was estimated by WST-1 assay. Morphological alterations were investigated with an inverted microscope. We investigated the effect of curcumin on ERAD and estrogen signaling proteins by immunoblotting assay. To evaluate the impact of curcumin on endoplasmic reticulum (ER) protein quality-related, the expression level of 32 genes was analyzed by quantitative reverse transcription polymerase chain reaction. The nuclear translocation of estrogen receptor was examined by nuclear fractionation and immunofluorescence microscopy. We found that curcumin effectively reduced the proliferation rates of LNCaP and VCaP cells. ERAD proteins; Hrd1, gp78, p97/VCP, Ufd1 and Npl4 were strongly induced by curcumin. Also, the steady-state level of polyubiquitin was increased in a dose-dependent manner in both cell lines. Curcumin administration remarkably decreased the protein levels of estrogen receptor-alfa (Erα), whereas estrogen receptor-beta unaffected. Additionally, curcumin strongly restricted the nuclear translocation of Erα. Present data suggest that curcumin may be effectively used in therapeutic approaches associated with the targeting ER protein quality control mechanism and modulation of estrogen signaling in prostate cancer.

TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein

Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determined that TTC17 is a large, soluble, ER localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and co-chaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of IGF1R, GPNMB, CLU and UGGT1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant

The loss of proteostasis due to reduced efficiency of protein degradation pathways plays a key role in multiple age-related diseases and is a hallmark of the aging process. Paradoxically, we have previously reported that the Caenorhabditis elegans rpn-10(ok1865) mutant, which lacks the RPN-10/RPN10/PSMD4 subunit of the 19S regulatory particle of the 26S proteasome, exhibits enhanced cytosolic proteostasis, elevated stress resistance and extended lifespan, despite possessing reduced proteasome function. However, the response of this mutant against threats to endoplasmic reticulum (ER) homeostasis and proteostasis was unknown. Here, we find that the rpn-10 mutant is highly ER stress resistant compared to the wildtype. Under unstressed conditions, the ER unfolded protein response (UPR) is activated in the rpn-10 mutant as signified by increased xbp-1 splicing. This primed response appears to alter ER homeostasis through the upregulated expression of genes involved in ER protein quality control (ERQC), including those in the ER-associated protein degradation (ERAD) pathway. Pertinently, we find that ERQC is critical for the rpn-10 mutant longevity. These changes also alter ER proteostasis, as studied using the C. elegans alpha-1 antitrypsin (AAT) deficiency model, which comprises an intestinal ER-localised transgenic reporter of an aggregation-prone form of AAT called ATZ. The rpn-10 mutant shows a significant reduction in the accumulation of the ATZ reporter, thus indicating that its ER proteostasis is augmented. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we then identified ecps-2/H04D03.3, a novel ortholog of the proteasome-associated adaptor and scaffold protein ECM29/ECPAS. We further show that ecps-2 is required for improved ER proteostasis as well as lifespan extension of the rpn-10 mutant. Thus, we propose that ECPS-2-proteasome functional interactions, alongside additional putative molecular processes, contribute to a novel ERQC adaptation which underlies the superior proteostasis and longevity of the rpn-10 mutant.

8-OR: Novel Mechanism of Lipoprotein Lipase Folding and Maturation in Adipocytes

Lipoprotein lipase (LPL) plays a central role in systemic lipid clearance by catalyzing intravascular hydrolysis of lipoprotein triglycerides, hence representing an important target in the treatment of hyperlipidemia in diabetes. Nascent LPL protein undergoes folding and maturation in the endoplasmic reticulum (ER), with only the properly folded ones exiting the ER and reaching to the cell surface. However, the maturation process for nascent LPL in the ER remains largely unknown. Here we show that LPL maturation in the ER is controlled by compensatory actions of two ER protein quality control mechanisms, ER-associated degradation (ERAD) and ER-phagy, in adipocytes. Specifically, we show that LPL is misfolding prone and degraded by ERAD, and when ERAD is compromised, LPL forms aggregates which is cleared by ER-phagy. In adipocytes lacking both ERAD and ER-phagy, ER fragments containing LPL aggregates cluster and fuse to form a novel cellular architecture, termed as Coalescence of ER Fragments (CERFs), where LPL undergo soluble-to-insoluble phase transition catalyzed by disulfide bond-mediated interactions. Lastly, mice with adipocyte specific ERAD and ER-phagy deficiency exhibited postprandial hyperlipidemia. In summary, this study provides novel insights into not only LPL biogenesis, but also the crosstalk between protein quality control pathways in the maintenance of ER homeostasis, as well as the disease pathogenesis, more broadly, associated with protein folding defects in the ER. Disclosure S.Wu: None. L.Qi: None.

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.

HERPUD1, a Member of the Endoplasmic Reticulum Protein Quality Control Mechanism, may be a Good Target for Suppressing Tumorigenesis in Breast Cancer Cells.

Breast cancer is the most frequently diagnosed cancer type and the second leading cause of cancer-related death in women. Recent studies have highlighted the importance of the endoplasmic reticulum (ER) protein quality control mechanism for the survival of many cancers. It has also been recommended as a good target for the treatment of many cancer types. Homocysteine inducible ER protein with ubiquitin-like domain 1 (HERPUD1) functions as one of the main components of ER-associated degradation, which is an ER-resident protein quality mechanism. Today, the association of HERPUD1 with breast carcinogenesis is still not fully understood. Herein, we evaluated the possibility of HERPUD1 as a potential therapeutic target for breast cancer. The effects of HERPUD1 silencing on epithelial-mesenchymal transition (EMT), angiogenesis, and cell cycle proteins were analyzed by immunoblotting studies. To test the role of HERPUD1 on tumorigenic features, WST-1-based cell proliferation assay, wound-healing assay, 2D colony formation assay, and Boyden-Chamber invasion assay were performed in human breast cancer cell line MCF-7. The statistical significance of the differences between the groups was determined by Student's t-test. Our results displayed that suppressing HERPUD1 expression reduced the cell cycle-related protein levels, including cyclin A2, cyclin B1, and cyclin E1 in MCF-7 cells. Also, silencing of HERPUD1 remarkably decreased expression levels of EMT-related N-cadherin and angiogenesis marker vascular endothelial growth factor A. Moreover, we determined that cell proliferation, migration, invasion, and colony formation of MCF-7 cells were significantly limited by silencing of HERPUD1. Present data suggest that HERPUD1 may be an effective target for biotechnological and pharmacological strategies to be developed to treat breast cancer.

Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside.

Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases.

Quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying recessive Ryr1 mutations linked to congenital myopathies.

Skeletal muscles are a highly structured tissue responsible for movement and metabolic regulation, which can be broadly subdivided into fast and slow twitch muscles with each type expressing common as well as specific sets of proteins. Congenital myopathies are a group of muscle diseases leading to a weak muscle phenotype caused by mutations in a number of genes including RYR1. Patients carrying recessive RYR1 mutations usually present from birth and are generally more severely affected, showing preferential involvement of fast twitch muscles as well as extraocular and facial muscles. In order to gain more insight into the pathophysiology of recessive RYR1-congential myopathies, we performed relative and absolute quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying p.Q1970fsX16 and p.A4329D RyR1 mutations which were identified in a child with a severe congenital myopathy. Our in-depth proteomic analysis shows that recessive RYR1 mutations not only decrease the content of RyR1 protein in muscle, but change the expression of 1130, 753, and 967 proteins EDL, soleus and extraocular muscles, respectively. Specifically, recessive RYR1 mutations affect the expression level of proteins involved in calcium signaling, extracellular matrix, metabolism and ER protein quality control. This study also reveals the stoichiometry of major proteins involved in excitation contraction coupling and identifies novel potential pharmacological targets to treat RyR1-related congenital myopathies.

The Activation of Reticulophagy by ER Stress through the ATF4-MAP1LC3A-CCPG1 Pathway in Ovarian Granulosa Cells Is Linked to Apoptosis and Necroptosis.

Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The ER unfolded protein response (UPRer) is initiated to promote cell survival by alleviating excessive ER stress, but cellular apoptosis is induced by persistent or strong ER stress. Recent studies have reported that reticulophagy is initiated by ER stress. Whether reticulophagy is activated in the ER stress-mediated apoptosis of granulosa cells and which pathway is initiated to activate reticulophagy during the apoptosis of granulosa cells are unknown. Therefore, the role of reticulophagy in granulosa cell death and the relationship between ER stress and reticulophagy were investigated in this work. Our results suggest that the ER stress inducer tunicamycin causes POF in mice, which is attributed to the apoptosis of granulosa cells and is accompanied by the activation of UPRer and reticulophagy. Furthermore, granulosa cells were treated with tunicamycin, and granulosa cell apoptosis was triggered and increased the expression of UPRer and reticulophagy molecules. The expression of ATF4 was then downregulated by RNAi, which decreased the levels of autophagy and the reticulophagy receptor CCGP1. Furthermore, ATF4 targets MAP1LC3A, as revealed by the ChIP sequencing results, and co-IP results demonstrated that MAP1LC3A interacts with CCPG1. Therefore, reticulophagy was activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway to mitigate ER stress. Additionally, the role of reticulophagy in granulosa cells was investigated by the knockdown of CCPG1 with RNAi. Interestingly, only a small number of granulosa cells died by apoptosis, whereas the death of most granulosa cells occurred by necroptosis triggered by STAT1 and STAT3 to impair ER proteostasis and the ER protein quality control system UPRer. Taken together, the results indicate that the necroptosis of granulosa cells is triggered by up- and downregulating the reticulophagy receptor CCPG1 through STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL and that reticulophagy is activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway.

Expression of the Z Variant of α1-Antitrypsin Suppresses Hepatic Cholesterol Biosynthesis in Transgenic Zebrafish.

Individuals homozygous for the Pi*Z allele of SERPINA1 (ZAAT) are susceptible to lung disease due to insufficient α1-antitrypsin secretion into the circulation and may develop liver disease due to compromised protein folding that leads to inclusion body formation in the endoplasmic reticulum (ER) of hepatocytes. Transgenic zebrafish expressing human ZAAT show no signs of hepatic accumulation despite displaying serum insufficiency, suggesting the defect in ZAAT secretion occurs independently of its tendency to form inclusion bodies. In this study, proteomic, transcriptomic, and biochemical analysis provided evidence of suppressed Srebp2-mediated cholesterol biosynthesis in the liver of ZAAT-expressing zebrafish. To investigate the basis for this perturbation, CRISPR/Cas9 gene editing was used to manipulate ER protein quality control factors. Mutation of erlec1 resulted in a further suppression in the cholesterol biosynthesis pathway, confirming a role for this ER lectin in targeting misfolded ZAAT for ER-associated degradation (ERAD). Mutation of the two ER mannosidase homologs enhanced ZAAT secretion without inducing hepatic accumulation. These insights into hepatic ZAAT processing suggest potential therapeutic targets to improve secretion and alleviate serum insufficiency in this form of the α1-antitrypsin disease.

Integration of ER protein quality control mechanisms defines β cell function and ER architecture.

Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β cells. SEL1L-HRD1 ERAD deficiency in β cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and β cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and β cell function.

Macro-ER-phagy receptors Atg39p and Atg40p confer resistance to aminoglycoside hygromycin B in S. cerevisiae.

Receptor-mediated autophagic turnover of portions of the endoplasmic reticulum (ER) is mediated by macro-ER-phagy. We hypothesized macro-ER-phagy promotes proteotoxic stress resistance. We predicted Saccharomyces cerevisiae lacking macro-ER-phagy receptors would exhibit enhanced sensitivity to hygromycin B, which reduces translational fidelity and is expected to globally disrupt protein homeostasis, including at the ER. We observed that loss of either of two yeast macro-ER-phagy receptors (Atg39p or Atg40p) compromised cellular resistance to hygromycin B to a similar extent as loss of ER-associated degradation (ERAD) ubiquitin ligases Hrd1p and Doa10p. Our data are consistent with a model whereby macro-ER-phagy and ERAD collaborate to mediate ER protein quality control. Disruptions of macro-ER-phagy have been linked to neuropathy, dementia, and cancer. A dampened capacity to mediate protein quality control may contribute to these conditions.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

Proofreading of protein localization mediated by a mitochondrial AAA-ATPase Msp1.

Normal cellular functions rely on correct protein localization within cells. Protein targeting had been thought to be a precise process, and even if it fails, the mistargeted proteins were supposed to be quickly degraded. However, this view is rapidly changing. Tail-anchored (TA) proteins are a class of membrane proteins that possess a single transmembrane domain (TMD) near the C-terminus and are posttranslationally targeted to the endoplasmic reticulum (ER) membrane, mitochondrial outer membrane (OM), and peroxisomal membrane, yet they can be mistargeted to the mitochondrial OM. The mistargeted TA proteins can be extracted from the OM by a mitochondrial AAA-ATPase Msp1/ATAD1 and transferred to the ER. If they are regarded as aberrant by the ER protein quality control system, they are extracted from the ER membrane for proteasomal degradation in the cytosol. If they are not regarded as aberrant, they are further transported to downstream organelles or original destinations along the secretory pathway. Thus, Msp1 contributes to not only degradation but also "proofreading" of the targeting of mislocalized TA proteins.

The Role of the Rhomboid Superfamily in ER Protein Quality Control: From Mechanisms and Functions to Diseases.

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells and is a major site for protein folding, modification, and lipid synthesis. Perturbations within the ER, such as protein misfolding and high demand for protein folding, lead to dysregulation of the ER protein quality control network and ER stress. Recently, the rhomboid superfamily has emerged as a critical player in ER protein quality control because it has diverse cellular functions, including ER-associated degradation (ERAD), endosome Golgi-associated degradation (EGAD), and ER preemptive quality control (ERpQC). This breadth of function both illustrates the importance of the rhomboid superfamily in health and diseases and emphasizes the necessity of understanding their mechanisms of action. Because dysregulation of rhomboid proteins has been implicated in various diseases, such as neurological disorders and cancers, they represent promising potential therapeutic drug targets. This review provides a comprehensive account of the various roles of rhomboid proteins in the context of ER protein quality control and discusses their significance in health and disease.