Endoplasmic Reticulum Quality Control System Research Articles

Regulation and function of endoplasmic reticulum autophagy in neurodegenerative diseases

The endoplasmic reticulum, a key cellular organelle, regulates a wide variety of cellular activities. Endoplasmic reticulum autophagy, one of the quality control systems of the endoplasmic reticulum, plays a pivotal role in maintaining endoplasmic reticulum homeostasis by controlling endoplasmic reticulum turnover, remodeling, and proteostasis. In this review, we briefly describe the endoplasmic reticulum quality control system, and subsequently focus on the role of endoplasmic reticulum autophagy, emphasizing the spatial and temporal mechanisms underlying the regulation of endoplasmic reticulum autophagy according to cellular requirements. We also summarize the evidence relating to how defective or abnormal endoplasmic reticulum autophagy contributes to the pathogenesis of neurodegenerative diseases. In summary, this review highlights the mechanisms associated with the regulation of endoplasmic reticulum autophagy and how they influence the pathophysiology of degenerative nerve disorders. This review would help researchers to understand the roles and regulatory mechanisms of endoplasmic reticulum-phagy in neurodegenerative disorders.

Structural and functional analysis of aquaporin-2 mutants involved in nephrogenic diabetes insipidus

Aquaporins are water channels found in the cell membrane, where they allow the passage of water molecules in and out of the cells. In the kidney collecting duct, arginine vasopressin-dependent trafficking of aquaporin-2 (AQP2) fine-tunes reabsorption of water from pre-urine, allowing precise regulation of the final urine volume. Point mutations in the gene for AQP2 may disturb this process and lead to nephrogenic diabetes insipidus (NDI), whereby patients void large volumes of highly hypo-osmotic urine. In recessive NDI, mutants of AQP2 are retained in the endoplasmic reticulum due to misfolding. Here we describe the structural and functional characterization of three AQP2 mutations associated with recessive NDI: T125M and T126M, situated close to a glycosylation site and A147T in the transmembrane region. Using a proteoliposome assay, we show that all three mutants permit the transport of water. The crystal structures of T125M and T126M together with biophysical characterization of all three mutants support that they retain the native structure, but that there is a significant destabilization of A147T. Our work provides unique molecular insights into the mechanisms behind recessive NDI as well as deepens our understanding of how misfolded proteins are recognized by the ER quality control system.

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.

Circadian Oscillation Pattern of Endoplasmic Reticulum Quality Control (ERQC) Components in Human Embryonic Kidney HEK293 Cells.

The circadian clock regulates the "push-pull" of the molecular signaling mechanisms that arrange the rhythmic organization of the physiology to maintain cellular homeostasis. In mammals, molecular clock genes tightly arrange cellular rhythmicity. It has been shown that this circadian clock optimizes various biological processes, including the cell cycle and autophagy. Hence, we explored the dynamic crosstalks between the circadian rhythm and endoplasmic reticulum (ER)-quality control (ERQC) mechanisms. ER-associated degradation (ERAD) is one of the most important parts of the ERQC system and is an elaborate surveillance system that eliminates misfolded proteins. It regulates the steady-state levels of several physiologically crucial proteins, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and the metastasis suppressor KAI1/CD82. However, the circadian oscillation of ERQC members and their roles in cellular rhythmicity requires further investigation. In the present study, we provided a thorough investigation of the circadian rhythmicity of the fifteen crucial ERQC members, including gp78, Hrd1, p97/VCP, SVIP, Derlin1, Ufd1, Npl4, EDEM1, OS9, XTP3B, Sel1L, Ufd2, YOD1, VCIP135 and FAM8A1 in HEK293 cells. We found that mRNA and protein accumulation of the ubiquitin conjugation, binding and processing factors, retrotranslocation-dislocation, substrate recognition and targeting components of ERQC exhibit oscillation under the control of the circadian clock. Moreover, we found that Hrd1 and gp78 have a possible regulatory function on Bmal1 turnover. The findings of the current study indicated that the expression level of ERQC components is fine-tuned by the circadian clock and major ERAD E3 ligases, Hrd1 and gp78, may influence the regulation of circadian oscillation by modulation of Bmal1 stability.

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris.

The industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability. Wild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed. The coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability. These findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

The functional specificity of ERECTA-family receptors in Arabidopsis stomatal development is ensured by molecular chaperones in the endoplasmic reticulum.

Stomata are epidermal pores that control gas exchange between plants and the atmosphere. In Arabidopsis, the ERECTA family (ERECTAf) receptors, including ERECTA, ERECTA-LIKE 1 (ERL1) and ERL2, redundantly play pivotal roles in enforcing the 'one-cell-spacing' rule. Accumulating evidence has demonstrated that the functional specificities of receptors are likely associated with their differential subcellular dynamics. The endoplasmic reticulum (ER)-resident chaperone complex SDF2-ERdj3B-BiP functions in many aspects of plant development. We employed pharmacological treatments combined with cell biological and biochemical approaches to demonstrate that the abundance of ERECTA was reduced in the erdj3b-1 mutant, but the localization and dynamics of ERECTA were not noticeably affected. By contrast, the erdj3b mutation caused the retention of ERL1/ERL2 in the ER. Furthermore, we found that the function of SDF2-ERdj3B-BiP is implicated with the distinct roles of ERECTAf receptors. Our findings establish that the ERECTAf receptor-mediated signaling in stomatal development is ensured by the activities of the ER quality control system, which preferentially maintains the protein abundance of ERECTA and proper subcellular dynamics of ERL1/ERL2, prior to the receptors reaching their destination - the plasma membrane - to execute their functions.

The Impact of Glycoengineering on the Endoplasmic Reticulum Quality Control System in Yeasts.

Yeasts are widely used and established production hosts for biopharmaceuticals. Despite of tremendous advances on creating human-type N-glycosylation, N-glycosylated biopharmaceuticals manufactured with yeasts are missing on the market. The N-linked glycans fulfill several purposes. They are essential for the properties of the final protein product for example modulating half-lives or interactions with cellular components. Still, while the protein is being formed in the endoplasmic reticulum, specific glycan intermediates play crucial roles in the folding of or disposal of proteins which failed to fold. Despite of this intricate interplay between glycan intermediates and the cellular machinery, many of the glycoengineering approaches are based on modifications of the N-glycan processing steps in the endoplasmic reticulum (ER). These N-glycans deviate from the canonical structures required for interactions with the lectins of the ER quality control system. In this review we provide a concise overview on the N-glycan biosynthesis, glycan-dependent protein folding and quality control systems and the wide array glycoengineering approaches. Furthermore, we discuss how the current glycoengineering approaches partially or fully by-pass glycan-dependent protein folding mechanisms or create structures that mimic the glycan epitope required for ER associated protein degradation.

Tandem mass tag-based proteomic analysis of endoplasmic reticulum proteins in mulberry leaves under ultraviolet-B and dark stress.

Mulberry leaves have been used in traditional Chinese medicine due to their antioxidant, antidiabetic, and antihyperlipidemic properties. A previous study showed that ultraviolet-B radiation followed by dark incubation could improve the contents of active ingredients in mulberry leaves, such as moracin N and chalcomoracin. The endoplasmic reticulum (ER) serves as a protein quality control center and the location for protein synthesis, which is involved in the response to the environmental stress in plants. To investigate the mechanisms in response to ultraviolet-B radiation followed by dark incubation (UV + D), ER proteomics was performed on mulberry leaves. The ER protein markers, glucose-regulated protein (GRP78), and calnexin (CNX), were significantly higher in the ER fraction than in the total protein fraction, indicating that the ER was purified. Compared to the control, the abundance of protein disulfide isomerase, UDP-glucose glycoprotein glucosyltransferase, CNX, and calreticulin proteins decreased, while of the abundance of heat shock-related proteins increased under stress. P450 enzyme system-related proteins and ribosomal proteins showed significant increases. These results suggest that under UV + D stress, mulberry leaves activated the cell redox and ER quality control systems, enhancing protein synthesis and weakening N-glycan biosynthesis in the ER to resist the damage.

A slowly cleaved viral signal peptide acts as a protein-integral immune evasion domain

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.

Identification of Specific Trafficking Defects of Naturally Occurring Variants of the Human ABCG2 Transporter.

Proper targeting of the urate and xenobiotic transporter ATP-binding transporter subfamily G member 2 (ABCG2) to the plasma membrane (PM) is essential for its normal function. The naturally occurring Q141K and M71V polymorphisms in ABCG2, associated with gout and hyperuricemia, affect the cellular routing of the transporter, rather than its transport function. The cellular localization of ABCG2 variants was formerly studied by immunolabeling, which provides information only on the steady-state distribution of the protein, leaving the dynamics of its cellular routing unexplored. In the present study, we assessed in detail the trafficking of the wild-type, M71V-, and Q141K-ABCG2 variants from the endoplasmic reticulum (ER) to the cell surface using a dynamic approach, the so-called Retention Using Selective Hooks (RUSH) system. This method also allowed us to study the kinetics of glycosylation of these variants. We found that the fraction of Q141K- and M71V-ABCG2 that passes the ER quality control system is only partially targeted to the PM; a subfraction is immobile and retained in the ER. Surprisingly, the transit of these variants through the Golgi apparatus (either the appearance or the exit) was unaffected; however, their PM delivery beyond the Golgi was delayed. In addition to identifying the specific defects in the trafficking of these ABCG2 variants, our study provides a novel experimental tool for studying the effect of drugs that potentially promote the cell surface delivery of mutant or polymorphic ABCG2 variants with impaired trafficking.

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation–enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count–based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture–based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

Calnexin mediates the maturation of GPI-anchors through ER retention

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.

Direct relationship between increased expression and mistrafficking of the Charcot–Marie–Tooth–associated protein PMP22

Charcot-Marie-Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ∼1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third WT PMP22 allele. In rodent models overexpressing the PMP22 (peripheral myelin protein 22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum quality control system, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of WT or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells and PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mistrafficking of PMP22, which is likely a contributor to disease pathology and progression.

Endoplasmic reticulum‐associated degradation mediated by MoHrd1 and MoDer1 is pivotal for appressorium development and pathogenicity of Magnaporthe oryzae

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER); however, protein folding is error-prone, resulting in toxic protein aggregation and cause ER stress. Irreversibly misfolded proteins are subjected to ER-associated degradation (ERAD), modified by ubiquitination, and degraded by the 26S proteasome. The yeast ERAD ubiquitin ligase Hrd1p and multispanning membrane protein Der1p are involved in ubiquitination and transportation of the folding-defective proteins. Here, we performed functional characterization of MoHrd1 and MoDer1 and revealed that both of them are localized to the ER and are pivotal for ERAD substrate degradation and the ER stress response. MoHrd1 and MoDer1 are involved in hyphal growth, asexual reproduction, infection-related morphogenesis, protein secretion and pathogenicity of M. oryzae. Importantly, MoHrd1 and MoDer1 mediated conidial autophagic cell death and subsequent septin ring assembly at the appressorium pore, leading to abnormal appressorium development and loss of pathogenicity. In addition, deletion of MoHrd1 and MoDer1 activated the basal unfolded protein response (UPR) and autophagy, suggesting that crosstalk between ERAD and two other closely related mechanisms in ER quality control system (UPR and autophagy) governs the ER stress response. Our study indicates the importance of ERAD function in fungal development and pathogenesis of M. oryzae.

PERK‐ and ATF6‐dependent cell quality control mechanisms in response to chronic expression of an aberrantly trafficked mutant ABCA3 lipid transporter

RATIONALEATP binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in the pulmonary surfactant function. A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease. Two functional classes of clinical ABCA3 mutants, designated as Type I and Type II, have been identified where Type I mutations cause aberrant protein trafficking and Type II mutations impair the lipid pump. The proline substitution for leucine mutations at residues 101 (ABCA3L101P) and 982 (ABCA3L982P) are designated as a Type I mutations. We previously reported that whereas ABCA3L101P is retained within the ER, ABCA3L982P partially escapes the ER quality control system and is localized in Rab 7‐positive, endosome/amphisome compartments, both mutations disrupting cellular homeostasis. The mechanism underlying cellular responses to chronic expression of these aberrantly trafficked transporters is incompletely defined.APPROACHIn vitro cell line models transiently or stably expressing fluorescent‐tagged ABCA3 negative controls (wild type and a functional mutant (ABCA3E292V)), positive control (ABCA3L101P) and experimental ABCA3L982P mutant isoforms were used for co‐localization confocal microscopy studies with subcellular organelle markers. Additionally, biochemical analyses of the cellular responses to acute and chronic expression of these mutant transporters were evaluated.RESULTSA549 and HEK293 cells transiently expressing ABCA3 isoforms showed clearance of overexpressed transporters via both the proteasome and autophagic pathways regardless of isoform (WT or mutants) or cell type. However, ER stress response (GRP78 and HDJ2) and activation of one of the three arms of the unfolded proteins response, IRE1 (upregulation of XBP1), were observed only in cells transiently expressing the mistargeted transporters (ABCA3L101P or ABCA3L982P) while ATF6 and PERK remained unaffected. The effect of long‐term expression of mistargeted mutant transporters on cell phenotype was evaluated using HEK293 cells stably expressing ABCA3L101P or ABCA3L982P. Compared to controls, chronic expression of the misstrafficked transporters elicited considerable PERK upregulation but normalization of IRE1 signaling. Likewise, significant ATF6 upregulation was observed only in cells expressing ABCA3L982P. Moreover, upregulation of autophagic flux was noted in cells expressing either trafficking mutant marked by substantial increases in LC3 and P62 levels during bafilomycin‐induced autophagic block. However, while steady state levels of the autophagic markers were increased in ABCA3L101P expressing cells, they were decreased in Rab7‐routed ABCA3L982P expressing cells (including Rab7).CONCLUSIONThese results indicate that cellular response and survival from long‐term expression of aberrantly trafficked ABCA3 mutant transporters is partially dependent on their subcellular routing but requires the adaptive and descriminatory use of overlapping proteostatic repertoires including upregulation of selective arms of the unfolded protein response and cellular macroautophagy.Support or Funding InformationNIH HL 129150 (SM)

Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae.

Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.

Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.

Folded or Degraded in Endoplasmic Reticulum.

In consistent with other membrane-bound and secretory proteins, immune checkpoint proteins go through a set of modifications in the endoplasmic reticulum (ER) to acquire their native functional structures before they function at their destinations. There are various ER-resident chaperones and enzymes synergistically regulate and catalyze the glycosylation, folding and transporting of proteins. The whole processing is under the surveillance of ER quality control system which allows the correctly folded proteins to exit from the ER with the help of coat proteinII(COPII) coated vesicles, while retains the rest of terminally misfolded ones in the ER and then eliminates them via ER-associated degradation (ERAD) or ER-to-lysosomes-associated degradation (ERLAD). The dysfunction of the ER causes ER stress which triggers unfolded protein response (UPR) to restore ER proteostasis. Unsolvable prolonged ER stress ultimately results in cell death. This chapter reviews the process that proteins undergo in the ER, and the glycosylation, folding and degradation of immune checkpoint proteins as well as the associated potential immunotherapies to date.

Disposing of misfolded ER proteins: A troubled substrate's way out of the ER

Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Those that fail must be recognized and eliminated to maintain ER homeostasis. Two cellular mechanisms have been identified to rid the ER of terminally unfolded, misfolded, and aggregated proteins. ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. ER-phagy has been more recently described and caters to larger, more complex proteins and protein aggregates that are not readily handled by ERAD. This pathway has unique upstream components and relies on the same downstream effectors of autophagy used in other cellular processes to deliver clients to lysosomes for degradation. In this review, we describe the main elements of ERQC, ERAD, and ER-phagy and focus on recent advances in these fields.

Endoplasmic reticulum quality control in lipoprotein metabolism

Lipids play a critical role in energy metabolism, and a suite of proteins is required to deliver lipids to tissues. Several of these proteins require an intricate endoplasmic reticulum (ER) quality control (QC) system and unique secondary chaperones for folding. Key examples include apolipoprotein B (apoB), which is the primary scaffold for many lipoproteins, dimeric lipases, which hydrolyze triglycerides from circulating lipoproteins, and the low-density lipoprotein receptor (LDLR), which clears cholesterol-rich lipoproteins from the circulation. ApoB requires specialized proteins for lipidation, dimeric lipases lipoprotein lipase (LPL) and hepatic lipase (HL) require a transmembrane maturation factor for secretion, and the LDLR requires several specialized, domain-specific chaperones. Deleterious mutations in these proteins or their chaperones may result in dyslipidemias, which are detrimental to human health. Here, we review the ER quality control systems that ensure secretion of apoB, LPL, HL, and LDLR with a focus on the specialized chaperones required by each protein.