ER chaperones use a protein folding and quality control glyco-code.

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.

Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Calnexin reveals a sugar-free taste within the lipid bilayer.

Maturation of membrane proteins is complicated by the need to fold in three distinct environments. While much is known about folding in the two aqueous milieus constituted by cytoplasm and ER lumen, our knowledge of the folding, arrangement, and quality control of transmembrane regions within the lipid bilayer, and its facilitation by molecular chaperones, is limited. New work by Bloemeke et al now reveals an expanded role of the ER chaperone calnexin acting within the lipid bilayer in a carbohydrate-independent manner.

Activating and Repressing IRE1α: The Hsp47 and BiP Tug of War.

In this issue of Molecular Cell, Sepulveda et al. (2018) discovered an interesting role of Hsp47 in regulating the unfolded protein response (UPR) wherein Hsp47 binds to IRE1α and displaces BiP, thereby activating the IRE1α arm of the UPR pathway by a previously undetermined mechanism.

Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy.

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

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 determine 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 cochaperones 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 insulin-like growth factor type 1 receptor, glycoprotein nonmetastatic melanoma protein B, clusterin, and UDP-glucose:glycoprotein glucosyltransferase 1 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.

Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences.

In this issue of Molecular Cell, Behnke et al. (2016) describe a novel cell-based peptide-binding assay and use it to analyze the binding specificities of the endoplasmic reticulum Hsp70 chaperone and its co-chaperones and to probe their different roles in protein quality control.

TPR-containing proteins control protein organization and homeostasis for the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.

The Role of Endoplasmic Reticulum Chaperones in Protein Folding and Quality Control.

Molecular chaperones assist the folding of nascent chains in the cell. Chaperones also aid in quality control decisions as persistent chaperone binding can help to sort terminal misfolded proteins for degradation. There are two major molecular chaperone families in the endoplasmic reticulum (ER) that assist proteins in reaching their native structure and evaluating the fidelity of the maturation process. The ER Hsp70 chaperone, BiP, supports adenine nucleotide-regulated binding to non-native proteins that possess exposed hydrophobic regions. In contrast, the carbohydrate-dependent chaperone system involving the membrane protein calnexin and its soluble paralogue calreticulin recognize a specific glycoform of an exposed hydrophilic protein modification for which the composition is controlled by a series of glycosidases and transferases. Here, we compare and contrast the properties, mechanisms of action and functions of these different chaperones systems that work in parallel, as well as together, to assist a large variety of substrates that traverse the eukaryotic secretory pathway.

Proper secretion of the serpin antithrombin relies strictly on thiol-dependent quality control.

The protein quality control machinery of the endoplasmic reticulum (ERQC) ensures that client proteins are properly folded. ERQC substrates may be recognized as nonnative by the presence of exposed hydrophobic surfaces, free thiols, or processed N-glycans. How these features dictate which ERQC pathways engage a given substrate is poorly understood. Here, using metabolic labeling, immunoprecipitations, various biochemical assays, and the human serpin antithrombin III (ATIII) as a model, we explored the role of ERQC systems in mammalian cells. Although ATIII has N-glycans and a hydrophobic core, we found that its quality control depended solely on free thiol content. Mutagenesis of all six Cys residues in ATIII to Ala resulted in its efficient secretion even though the product was not natively folded. ATIII variants with free thiols were retained in the endoplasmic reticulum but not degraded. These results provide insight into the hierarchy of ERQC systems and reveal a fundamental vulnerability of ERQC in a case of reliance on the thiol-dependent quality control pathway.

You got to know when to hold (or unfold) 'em .

In this issue of Molecular Cell, Hoffmann et al. (2012) demonstrate that the ribosome-associated bacterial chaperone Trigger Factor assists in the maturation of ribosome-attached nascent chains by acting as both a holdase and an unfoldase.

EDEM1's mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity.

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is a protein quality control factor that was initially proposed to recognize N-linked glycans on misfolded proteins through its mannosidase-like domain (MLD). However, recent studies have demonstrated that EDEM1 binds to some misfolded proteins in a glycan-independent manner, suggesting a more complex binding landscape for EDEM1. In this study, we have identified a thiol-dependent substrate interaction between EDEM1 and the α1-antitrypsin ER-associated protein degradation (ERAD) clients Z and NHK, specifically through the single Cys residue on Z/NHK (Cys256), required for binding under stringent detergent conditions. In addition to the thiol-dependent interaction, the presence of weaker protein-protein interactions was confirmed, suggestive of bipartite client-binding properties. About four reactive thiols on EDEM1 were identified and were not directly responsible for the observed redox-sensitive binding by EDEM1. Moreover, a protein construct comprising the EDEM1 MLD had thiol-dependent binding properties along with its active glycan-trimming activities. Lastly, we identified an additional intrinsically disordered region (IDR) located at the C terminus of EDEM1 in addition to its previously identified N-terminal IDR. We also determined that both IDRs are required for binding to the ERAD component ERdj5 as an interaction with ERdj5 was not observed with the MLD alone. Together, our findings indicate that EDEM1 employs different binding modalities to interact with ERAD clients and ER quality control (ERQC) machinery partners and that some of these properties are shared with its homologues EDEM2 and EDEM3.

Cellular folding pathway of a metastable serpin.

Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly ß-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the protease-binding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.

N-Glycan-based ER Molecular Chaperone and Protein Quality Control System: The Calnexin Binding Cycle.

Helenius and colleagues proposed over 20-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate-binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review.

N-linked sugar-regulated protein folding and quality control in the ER.

Asparagine-linked glycans (N-glycans) are displayed on the majority of proteins synthesized in the endoplasmic reticulum (ER). Removal of the outermost glucose residue recruits the lectin chaperone malectin possibly involved in a first triage of defective polypeptides. Removal of a second glucose promotes engagement of folding and quality control machineries built around the ER lectin chaperones calnexin (CNX) and calreticulin (CRT) and including oxidoreductases and peptidyl-prolyl isomerases. Deprivation of the last glucose residue dictates the release of N-glycosylated polypeptides from the lectin chaperones. Correctly folded proteins are authorized to leave the ER. Non-native polypeptides are recognized by the ER quality control key player UDP-glucose glycoprotein glucosyltransferase 1 (UGT1), re-glucosylated and re-addressed to the CNX/CRT chaperone binding cycle to provide additional opportunity for the protein to fold in the ER. Failure to attain the native structure determines the selection of the misfolded polypeptides for proteasome-mediated degradation.

The molecular dating game: an antibody heavy chain hangs loose with a chaperone while waiting for its life partner.

In a recent issue of Molecular Cell, Feige et al. (2009) utilize the murine immunoglobulin system to shed light on a long-standing puzzle: how do cells coordinate folding of different polypeptides that ultimately form a complex?

EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex.

Terminally misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently cleared by the ER-associated degradation (ERAD) pathway. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however, the mechanisms of substrate recognition and sorting to the ERAD pathway are poorly defined. EDEM1 (ER degradation-enhancing alpha-mannosidase-like 1 protein) has been proposed to play a role in ERAD substrate signaling or recognition. We show that EDEM1 specifically binds nonnative proteins in a glycan-independent manner. Inhibition of mannosidase activity with kifunensine or disruption of the EDEM1 mannosidase-like domain by mutation had no effect on EDEM1 substrate binding but diminished its association with the ER membrane adaptor protein SEL1L. These results support a model whereby EDEM1 binds nonnative proteins and uses its mannosidase-like domain to target aberrant proteins to the ER membrane dislocation and ubiquitination complex containing SEL1L.

Flagging and docking: dual roles for N-glycans in protein quality control and cellular proteostasis.

Nascent polypeptides entering the endoplasmic reticulum (ER) are covalently modified with pre-assembled oligosaccharides. The terminal glucose and mannose residues are immediately removed after transfer of the oligosaccharide onto newly synthesized polypeptides. This processing determines whether the polypeptide will be retained in the ER, transported along the secretory pathway, or dislocated across the ER membrane for destruction. New avenues of research and some issues of controversy have recently been opened by the discovery that lectin-oligosaccharide interactions stabilize supramolecular complexes between regulators of ER-associated degradation (ERAD). In this Opinion article, we propose a unified model that depicts carbohydrates acting both as flags signaling the fitness of a maturing protein and as docking sites that regulate the assembly and stability of the ERAD machinery.

The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis.

Proteins that traffic through the eukaryotic secretory pathway are commonly modified with N-linked carbohydrates. These bulky amphipathic modifications at asparagines intrinsically enhance solubility and folding energetics through carbohydrate-protein interactions. N-linked glycans can also extrinsically enhance glycoprotein folding by using the glycoprotein homeostasis or 'glycoproteostasis' network, which comprises numerous glycan binding and/or modification enzymes or proteins that synthesize, transfer, sculpt and use N-linked glycans to direct folding and trafficking versus degradation and trafficking of nascent N-glycoproteins through the cellular secretory pathway. If protein maturation is perturbed by misfolding, aggregation or both, stress pathways are often activated that result in transcriptional remodeling of the secretory pathway in an attempt to alleviate the insult (or insults). The inability to achieve glycoproteostasis is linked to several pathologies, including amyloidoses, cystic fibrosis and lysosomal storage diseases. Recent progress on genetic and pharmacologic adaptation of the glycoproteostasis network provides hope that drugs of this mechanistic class can be developed for these maladies in the near future.

TMTC1 and TMTC2 are novel endoplasmic reticulum tetratricopeptide repeat-containing adapter proteins involved in calcium homeostasis.

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.