Reshaping endoplasmic reticulum quality control through the unfolded protein response.

Endoplasmic reticulum quality control (ERQC) pathways comprising chaperones, folding enzymes, and degradation factors ensure the fidelity of ER protein folding and trafficking to downstream secretory environments. However, multiple factors, including tissue-specific secretory proteomes, environmental and genetic insults, and organismal aging, challenge ERQC. Thus, a key question is: how do cells adapt ERQC to match the diverse, ever-changing demands encountered during normal physiology and in disease? The answer lies in the unfolded protein response (UPR), a signaling mechanism activated by ER stress. In mammals, the UPR comprises three signaling pathways regulated downstream of the ER membrane proteins IRE1, ATF6, and PERK. Upon activation, these UPR pathways remodel ERQC to alleviate cellular stress and restore ER function. Here, we describe how UPR signaling pathways adapt ERQC, highlighting their importance for maintaining ER function across tissues and the potential for targeting the UPR to mitigate pathologies associated with protein misfolding diseases.

Members of the Hsp70 Family Recognize Distinct Types of Sequences to Execute ER Quality Control.

Protein maturation in the endoplasmic reticulum is controlled by multiple chaperones, but how they recognize and determine the fate of their clients remains unclear. We developed an in vivo peptide library covering substrates of the ER Hsp70 system: BiP, Grp170, and three of BiP's DnaJ-family co-factors (ERdj3, ERdj4, and ERdj5). In vivo binding studies revealed that sites for pro-folding chaperones BiP and ERdj3 were frequent and dispersed throughout the clients, whereas Grp170, ERdj4, and ERdj5 specifically recognized a distinct type of rarer sequence with a high predicted aggregation potential. Mutational analyses provided insights into sequence recognition characteristics for these pro-degradation chaperones, which could be readily introduced or disrupted, allowing the consequences for client fates to be determined. Our data reveal unanticipated diversity in recognition sequences for chaperones; establish a sequence-encoded interplay between protein folding, aggregation, and degradation; and highlight the ability of clients to co-evolve with chaperones, ensuring quality control.

The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis.

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.

Acidification activates ERp44--a molecular litmus test for protein assembly.

In this issue of Molecular Cell, Vavassori et al. (2013) show that a pH-induced conformational change in the quality control protein ERp44 allows retrieval of secretory proteins that contain free thiols via a disulfide linkage from postendoplasmic reticulum compartments to prevent their premature secretion.

Quality control of integral membrane proteins by assembly-dependent membrane integration.

Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αßT cell receptor (αßTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis.

Role of the HSP70 Co-Chaperone SIL1 in Health and Disease.

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1's function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1's functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1's NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.

The endoplasmic reticulum (ER) chaperone BiP is a master regulator of ER functions: Getting by with a little help from ERdj friends.

The endoplasmic reticulum (ER) represents the entry point into the secretory pathway where nascent proteins encounter a specialized environment for their folding and maturation. Inherent to these processes is a dedicated quality-control system that detects proteins that fail to mature properly and targets them for cytosolic degradation. An imbalance in protein folding and degradation can result in the accumulation of unfolded proteins in the ER, resulting in the activation of a signaling cascade that restores proper homeostasis in this organelle. The ER heat shock protein 70 (Hsp70) family member BiP is an ATP-dependent chaperone that plays a critical role in these processes. BiP interacts with specific ER-localized DnaJ family members (ERdjs), which stimulate BiP's ATP-dependent substrate interactions, with several ERdjs also binding directly to unfolded protein clients. Recent structural and biochemical studies have provided detailed insights into the allosteric regulation of client binding by BiP and have enhanced our understanding of how specific ERdjs enable BiP to perform its many functions in the ER. In this review, we discuss how BiP's functional cycle and interactions with ERdjs enable it to regulate protein homeostasis in the ER and ensure protein quality control.

Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids.

Any protein synthesized in the secretory pathway has the potential to misfold and would need to be recognized and ubiquitylated for degradation. This is astounding, since only a few ERAD-specific E3 ligases have been identified. To begin to understand substrate recognition, we wished to map the ubiquitylation sites on the NS-1 nonsecreted immunoglobulin light chain, which is an ERAD substrate. Ubiquitin is usually attached to lysine residues and less frequently to the N terminus of proteins. In addition, several viral E3s have been identified that attach ubiquitin to cysteine or serine/threonine residues. Mutation of lysines, serines, and threonines in the NS-1 variable region was necessary to significantly reduce ubiquitylation and stabilize the protein. The Hrd1 E3 ligase was required to modify all three amino acids. Our studies argue that ubiquitylation of ER proteins relies on very different mechanisms of recognition and modification than those used to regulate biological processes.

An unfolded CH1 domain controls the assembly and secretion of IgG antibodies.

A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the C(H)1 domain of the heavy chain is intrinsically disordered in vitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain C(L) domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the C(H)1 domain and competes for binding to the C(L) domain. In vivo experiments demonstrate that requirements identified for folding the C(H)1 domain in vitro, including association with a folded C(L) domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell.

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins.

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.

Dimerization-dependent folding underlies assembly control of the clonotypic αßT cell receptor chains.

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here we used the clonotypic αß chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ER quality control with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αßTCR assembly control in the cell. We found that folding of the TCR α chain constant domain Cα is dependent on αß heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/ß assembly process, which allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and, therefore, reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α and ß chain combinations that comprise the T cell repertoire.

The large Hsp70 Grp170 binds to unfolded protein substrates in vivo with a regulation distinct from conventional Hsp70s.

The Hsp70 superfamily is a ubiquitous chaperone class that includes conventional and large Hsp70s. BiP is the only conventional Hsp70 in the endoplasmic reticulum (ER) whose functions include: assisting protein folding, targeting misfolded proteins for degradation, and regulating the transducers of the unfolded protein response. The ER also possesses a single large Hsp70, the glucose-regulated protein of 170 kDa (Grp170). Like BiP it is an essential protein, but its cellular functions are not well understood. Here we show that Grp170 can bind directly to a variety of incompletely folded protein substrates in the ER, and as expected for a bona fide chaperone, it does not interact with folded secretory proteins. Our data demonstrate that Grp170 and BiP associate with similar molecular forms of two substrate proteins, but while BiP is released from unfolded substrates in the presence of ATP, Grp170 remains bound. In comparison to conventional Hsp70s, the large Hsp70s possess two unique structural features: an extended C-terminal α-helical domain and an unstructured loop in the putative substrate binding domain with an unknown function. We find that in the absence of the α-helical domain the interaction of Grp170 with substrates is reduced. In striking contrast, deletion of the unstructured loop results in increased binding to substrates, suggesting the presence of unique intramolecular mechanisms of control for the chaperone functions of large Hsp70s.

Endoplasmic reticulum (ER) stress and hypoxia response pathways interact to potentiate hypoxia-inducible factor 1 (HIF-1) transcriptional activity on targets like vascular endothelial growth factor (VEGF).

Cells respond to suboptimal microenvironments by activating stress signaling pathways, like the unfolded protein response and hypoxia-induced transcription factors HIF-1/2, to restore homeostasis. Both cytoprotective pathways have been well studied in isolation at the biochemical and molecular levels. Mounting evidence reveals that they can be activated simultaneously in tumor cells and, likely, in other tissues experiencing inadequate microenvironments and that they share some transcriptional targets, like the proangiogenic factor VEGFA. However, the potential interaction between these pathways is poorly understood. Cell culture experiments revealed that as a consequence of unfolded protein response activation, ATF4 bound to the human VEGFA promoter and activated its transcription, whereas HIF-1 did so in response to hypoxia. When both pathways were activated together, VEGFA transcripts were induced to a higher level than when either stress was applied alone. Surprisingly, this was not due to the combined actions of the stress pathway-specific transcription factors. Instead, we found that endoplasmic reticulum stress potentiated HIF-1 activity to transactivate VEGF expression as well as another well characterized target, BNIP3. These data reveal an unexpected interaction between two important cytoprotective responses that are likely to have significant consequences in environmentally compromised tissues and tumor cells.

Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.

Examination of a second node of translational control in the unfolded protein response.

The unfolded protein response (UPR) is a largely cytoprotective signaling cascade that acts to re-establish homeostasis of the endoplasmic reticulum (ER) under conditions of stress by inducing an early and transient block in general protein synthesis and by increasing the folding and degradative capacity of the cell through an extensive transcriptional program. It is well established that the mechanism for the early translational attenuation during ER stress occurs through phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by activated PERK. Our data demonstrate that when eIF2α is dephosphorylated translation is not fully restored to pre-stressed levels. We found that this correlates with reduced mTOR activity and as a result decreases phosphorylation of 4E-BP1, which negatively regulates assembly of the eIF4F complex and cap-dependent translation. The decrease in mTOR activity and 4E-BP1 phosphorylation is associated with activation of AMP kinase, a negative regulator of mTOR, and in the case of some stress conditions, downregulation of signaling through key components of the PI3K pathway. Furthermore, we show that there is a subset of mRNAs that does not recover from UPR-induced translational repression, including those whose translation is particularly sensitive to loss of eIF4F, such as cyclin D1, Bcl-2 and MMP-9. Together these data implicate reduced mTOR activity and 4E-BP1 hypophosphorylation as a second, more restricted mechanism of translational control occurring somewhat later in the UPR.

How antibodies fold.

B cells use unconventional strategies for the production of a seemingly unlimited number of antibodies from a very limited amount of DNA. These methods dramatically increase the likelihood of producing proteins that cannot fold or assemble appropriately. B cells are therefore particularly dependent on 'quality control' mechanisms to oversee antibody production. Recent in vitro experiments demonstrate that Ig domains have evolved diverse folding strategies ranging from robust spontaneous folding to intrinsically disordered domains that require assembly with their partner domains to fold; in vivo experiments reveal that these different folding characteristics form the basis for cellular checkpoints in Ig transport. Taken together, these reports provide a detailed understanding of how B cells monitor and ensure the functional fidelity of Ig proteins.

A shared endoplasmic reticulum-associated degradation pathway involving the EDEM1 protein for glycosylated and nonglycosylated proteins.

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF(Fbs2) E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.

Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy.

SIL1 (also called BAP) acts as a nucleotide exchange factor for the Hsp70 chaperone BiP (also called GRP78), which is a key regulator of the main functions of the endoplasmic reticulum. We found nine distinct mutations that would disrupt the SIL1 protein in individuals with Marinesco-Sjögren syndrome, an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy. Identification of SIL1 mutations implicates Marinesco-Sjögren syndrome as a disease of endoplasmic reticulum dysfunction and suggests a role for this organelle in multisystem disorders.

The Essential Functions of Molecular Chaperones and Folding Enzymes in Maintaining Endoplasmic Reticulum Homeostasis.

It has been estimated that up to one-third of the proteins encoded by the human genome enter the endoplasmic reticulum (ER) as extended polypeptide chains where they undergo covalent modifications, fold into their native structures, and assemble into oligomeric protein complexes. The fidelity of these processes is critical to support organellar, cellular, and organismal health, and is perhaps best underscored by the growing number of disease-causing mutations that reduce the fidelity of protein biogenesis in the ER. To meet demands encountered by the diverse protein clientele that mature in the ER, this organelle is populated with a cadre of molecular chaperones that prevent protein aggregation, facilitate protein disulfide isomerization, and lower the activation energy barrier of cis-trans prolyl isomerization. Components of the lectin (glycan-binding) chaperone system also reside within the ER and play numerous roles during protein biogenesis. In addition, the ER houses multiple homologs of select chaperones that can recognize and act upon diverse peptide signatures. Moreover, redundancy helps ensure that folding-compromised substrates are unable to overwhelm essential ER-resident chaperones and enzymes. In contrast, the ER in higher eukaryotic cells possesses a single member of the Hsp70, Hsp90, and Hsp110 chaperone families, even though several homologs of these molecules reside in the cytoplasm. In this review, we discuss specific functions of the many factors that maintain ER quality control, highlight some of their interactions, and describe the vulnerabilities that arise from the absence of multiple members of some chaperone families.