Endoplasmic Reticulum Proteostasis Network Research Articles

ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants.

The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired function on neuronal membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we investigated the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A subunit, including M705V and A727T. In the transfected HEK293T cells, we showed that the two variants were targeted to the proteasome for degradation and had reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (binding immunoglobulin protein) in the endoplasmic reticulum (ER), dose-dependently enhanced the functional surface expression of the M705V and A727T variants in HEK293T cells. Moreover, BIX (10 μM) increased the surface protein levels of the M705V variant in human iPSC-derived neurons. We revealed that BIX promoted folding, inhibited degradation, and enhanced anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results suggest that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope.

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

Mastering organismal aging through the endoplasmic reticulum proteostasis network.

The aging process is characterized by a progressive decline in the function of most tissues, representing the main risk factor in the development of a variety of human diseases. Studies in multiple animal models have demonstrated that interventions that improve the capacity to maintain endoplasmic reticulum (ER) proteostasis prolong life and healthspan. ER stress is monitored by the unfolded protein response (UPR), a signaling pathway that mediates adaptive processes to restore proteostasis or the elimination of damaged cells by apoptosis. Here, we discuss recent advances in understanding the significance of the UPR to aging and its implications for the maintenance of cell physiology of various cell types and organs. The possible benefits of targeting the UPR to extend healthspan and reduce the risk of developing age‐related diseases are also discussed.

ATF6 as a Nodal Regulator of Proteostasis in the Heart.

Proteostasis encompasses a homeostatic cellular network in all cells that maintains the integrity of the proteome, which is critical for optimal cellular function. The components of the proteostasis network include protein synthesis, folding, trafficking, and degradation. Cardiac myocytes have a specialized endoplasmic reticulum (ER) called the sarcoplasmic reticulum that is well known for its role in contractile calcium handling. However, less studied is the proteostasis network associated with the ER, which is of particular importance in cardiac myocytes because it ensures the integrity of proteins that are critical for cardiac contraction, e.g., ion channels, as well as proteins necessary for maintaining myocyte viability and interaction with other cell types, e.g., secreted hormones and growth factors. A major aspect of the ER proteostasis network is the ER unfolded protein response (UPR), which is initiated when misfolded proteins in the ER activate a group of three ER transmembrane proteins, one of which is the transcription factor, ATF6. Prior to studies in the heart, ATF6 had been shown in model cell lines to be primarily adaptive, exerting protective effects by inducing genes that encode ER proteins that fortify protein-folding in this organelle, thus establishing the canonical role for ATF6. Subsequent studies in isolated cardiac myocytes and in the myocardium, in vivo, have expanded roles for ATF6 beyond the canonical functions to include the induction of genes that encode proteins outside of the ER that do not have known functions that are obviously related to ER protein-folding. The identification of such non-canonical roles for ATF6, as well as findings that the gene programs induced by ATF6 differ depending on the stimulus, have piqued interest in further research on ATF6 as an adaptive effector in cardiac myocytes, underscoring the therapeutic potential of activating ATF6 in the heart. Moreover, discoveries of small molecule activators of ATF6 that adaptively affect the heart, as well as other organs, in vivo, have expanded the potential for development of ATF6-based therapeutics. This review focuses on the ATF6 arm of the ER UPR and its effects on the proteostasis network in the myocardium.

Mutation‐Dependent Capacity of the ER Proteostasis Network to Quality Control Disease‐Causing Collagen Misfolding

Osteogenesis imperfecta (OI) is an inherited disease most commonly caused by autosomal dominant mutations in collagen type‐I. OI‐causing mutations are found in both the triple helical region and the C‐propeptide region of collagen‐I. Mutations in the triple helical region cause disruption to triple helical folding, stability, and structure. Mutations in the C‐propeptide region, the C‐terminal nucleation domain of collagen‐I, disrupt the initial assembly steps in collagen folding. Currently, the mechanism by which the cellular protein folding machinery recognizes and processes these types of misfolded variants of collagen‐I is not well‐understood. Here, we report how the endoplasmic reticulum (ER) differentially processes normal and misfolding, OI‐causing collagen‐I variants. We employed state‐of‐the‐art, quantitative mass spectrometry‐based proteomic methods to determine the full complement of proteostasis network components that differentially engage wild‐type (wt) and several OI‐causing mutants of collagen‐I. We identified >75 putative proteins that might play a role in collagen‐I proteostasis, in addition to the currently known collagen‐I interactors. Interestingly, we found that while there is no significant difference in the interactomes of wild‐type (wt) and triple‐helical mutant collagen‐I variants, mutant C‐propeptide collagen‐I is well‐recognized by a number of components of the ER proteostasis network. These results indicate that the cell can specifically recognize collagen C‐propeptide misfolding, but has limited capacity to address triple‐helix domain misfolding. Our findings contribute to an understanding of how the proteostasis network reacts to different types of collagen folding defects and maintains proper collagen folding and secretion.

LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors

The endoplasmic reticulum-Golgi intermediate compartment protein-53 (ERGIC-53, aka LMAN1), which cycles between the endoplasmic reticulum (ER) and Golgi, is a known cargo receptor for a number of soluble proteins. However, whether LMAN1 plays a role as a trafficking factor in the central nervous system is largely unknown. Here, we determined the role of LMAN1 on endogenous protein levels of the Cys-loop superfamily of neuroreceptors, including gamma-aminobutyric acid type A receptors (GABAARs), 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and nicotinic acetylcholine receptors (nAChRs). Knockdown of LMAN1 reduces the surface trafficking of endogenous β3 subunits of GABAARs in mouse hypothalamic GT1-7 neurons. Furthermore, Western blot analysis of brain homogenates from LMAN1 knockout mice demonstrated that loss of LMAN1 decreases the total protein levels of 5HT3A receptors and γ2 subunits of GABAARs. LMAN1 knockout regulates the ER proteostasis network by upregulating ERP44 without changing calnexin levels. Interestingly, despite the critical role of the glycan-binding function of LMAN1 in its other known cargo clients, LMAN1 interacts with GABAARs in a glycan-independent manner. In summary, LMAN1 is a trafficking factor for certain neuroreceptors in the central nervous system. This is the first report of LMAN1 function in membrane protein trafficking.

Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.

Biogenesis of membrane proteins is controlled by the protein homeostasis (proteostasis) network. We have been focusing on protein quality control of γ-aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter-gated ion channels in mammalian central nervous system. Proteostasis deficiency in GABAA receptors causes loss of their surface expression and thus function on the plasma membrane, leading to epilepsy and other neurological diseases. One well-characterized example is the A322D mutation in the α1 subunit that causes its extensive misfolding and expedited degradation in the endoplasmic reticulum (ER), resulting in autosomal dominant juvenile myoclonic epilepsy. We aimed to correct misfolding of the α1(A322D) subunits in the ER as an approach to restore their functional surface expression. Here, we showed that application of BIX, a specific, potent ER resident HSP70 family protein BiP activator, significantly increases the surface expression of the mutant receptors in human HEK293T cells and neuronal SH-SY5Y cells. BIX attenuates the degradation of α1(A322D) and enhances their forward trafficking and function. Furthermore, because BiP is one major target of the two unfolded protein response (UPR) pathways: ATF6 and IRE1, we continued to demonstrate that modest activations of the ATF6 pathway and IRE1 pathway genetically enhance the plasma membrane trafficking of the α1(A322D) protein in HEK293T cells. Our results underlie the potential of regulating the ER proteostasis network to correct loss-of-function protein conformational diseases.

Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

Pharmacologic arm-selective unfolded protein response (UPR) signaling pathway activation is emerging as a promising strategy to ameliorate imbalances in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. The small molecule N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (147) was previously identified (<xref ref-type="bibr" rid="bib35">Plate et al., 2016</xref>) to preferentially activate the ATF6 arm of the UPR, promoting protective remodeling of the ER proteostasis network. Here we show that 147-dependent ATF6 activation requires metabolic oxidation to form an electrophile that preferentially reacts with ER proteins. Proteins covalently modified by 147 include protein disulfide isomerases (PDIs), known to regulate ATF6 activation. Genetic depletion of PDIs perturbs 147-dependent induction of the ATF6-target gene, BiP, implicating covalent modifications of PDIs in the preferential activation of ATF6 afforded by treatment with 147. Thus, 147 is a pro-drug that preferentially activates ATF6 signaling through a mechanism involving localized metabolic activation and selective covalent modification of ER resident proteins that regulate ATF6 activity.

Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

Stress-Independent Activation of XBP1s and/or ATF6 Reveals Three Functionally Diverse ER Proteostasis Environments

The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.