Metabolic and immune-sensitive contacts between lipid droplets and endoplasmic reticulum reconstituted in vitro.

Coordinated cell function requires a variety of subcellular organelles to exchange proteins and lipids across physical contacts that are also referred to as membrane contact sites. Such organelle-to-organelle contacts also evoke interest because they can appear in response to metabolic changes, immune activation, and possibly other stimuli. The microscopic size and complex, crowded geometry of these contacts, however, makes them difficult to visualize, manipulate, and understand inside cells. To address this shortcoming, we deposited endoplasmic reticulum (ER)-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a proteinaceous planar membrane. We visualized real-time lipid and protein exchange across contacts that form between this ER-mimicking membrane and lipid droplets (LDs) purified from the liver of rat. The high-throughput imaging possible in this geometry reveals that in vitro LD-ER contacts increase dramatically when the metabolic state is changed by feeding the animal and also when the immune system is activated. Contact formation in both cases requires Rab18 GTPase and phosphatidic acid, thus revealing common molecular targets operative in two very different biological pathways. An optical trap is used to demonstrate physical tethering of individual LDs to the ER-mimicking membrane and to estimate the strength of this tether. These methodologies can potentially be adapted to understand and target abnormal contact formation between different cellular organelles in the context of neurological and metabolic disorders or pathogen infection.

Gain-of-function genetic screening identifies the antiviral function of TMEM120A via STING activation.

Zika virus (ZIKV) infection can be associated with neurological pathologies, such as microcephaly in newborns and Guillain-Barre syndrome in adults. Effective therapeutics are currently not available. As such, a comprehensive understanding of virus-host interactions may guide the development of medications for ZIKV. Here we report a human genome-wide overexpression screen to identify host factors that regulate ZIKV infection and find TMEM120A as a ZIKV restriction factor. TMEM120A overexpression significantly inhibits ZIKV replication, while TMEM120A knockdown increases ZIKV infection in cell lines. Moreover, Tmem120a knockout in mice facilitates ZIKV infection in primary mouse embryonic fibroblasts (MEF) cells. Mechanistically, the antiviral activity of TMEM120A is dependent on STING, as TMEM120A interacts with STING, promotes the translocation of STING from the endoplasmic reticulum (ER) to ER-Golgi intermediate compartment (ERGIC) and enhances the phosphorylation of downstream TBK1 and IRF3, resulting in the expression of multiple antiviral cytokines and interferon-stimulated genes. In summary, our gain-of-function screening identifies TMEM120A as a key activator of the antiviral signaling of STING.

Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies.

Glucose-related protein 78 (GRP78) is a chaperone protein localized primarily in the endoplasmic reticulum (ER) lumen, where it helps in proper protein folding by targeting misfolded proteins and facilitating protein assembly. In stressed cells, GRP78 is translocated to the cell surface (csGRP78) where it binds to various ligands and triggers different intracellular pathways. Thus, csGRP78 expression is associated with cancer, involved in the maintenance and progression of the disease. Extracellular exposition of csGRP78 leads to the production of autoantibodies as observed in patients with prostate or ovarian cancer, in which the ability to target csGRP78 affects the tumor development. Present on the surface of cancer cells and not normal cells in vivo, csGRP78 represents an interesting target for therapeutic antibody strategies. Here we give an overview of the csGRP78 function in the cell and its role in oncogenesis, thereby providing insight into the clinical value of GRP78 monoclonal antibodies for cancer prognosis and treatment.

Mitochondrial aspartate regulates TNF biogenesis and autoimmune tissue inflammation.

Misdirected immunity gives rise to the autoimmune tissue inflammation of rheumatoid arthritis, in which excess production of the cytokine tumor necrosis factor (TNF) is a central pathogenic event. Mechanisms underlying the breakdown of self-tolerance are unclear, but T cells in the arthritic joint have a distinctive metabolic signature of ATPlo acetyl-CoAhi proinflammatory effector cells. Here we show that a deficiency in the production of mitochondrial aspartate is an important abnormality in these autoimmune T cells. Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide, causing ADP deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP. As a result, ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of transmembrane TNF. ERrich T cells were the predominant TNF producers in the arthritic joint. Transfer of intact mitochondria into T cells, as well as supplementation of exogenous aspartate, rescued the mitochondria-instructed expansion of ER membranes and suppressed TNF release and rheumatoid tissue inflammation.

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism.

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

Neuromyelitis optica (NMO)-IgG-driven organelle reorganization in human iPSC-derived astrocytes.

Neuromyelitis optica (NMO) is an autoimmune disease that primarily targets astrocytes. Autoantibodies (NMO-IgG) against the water channel protein, aquaporin 4 (AQP4), are a serologic marker in NMO patients, and they are known to be responsible for the pathophysiology of the disease. In the brain, AQP4 is mainly expressed in astrocytes, especially at the end-feet, where they form the blood-brain barrier. Following the interaction between NMO-IgG and AQP4 in astrocytes, rapid AQP4 endocytosis initiates pathogenesis. However, the cellular and molecular mechanisms of astrocyte destruction by autoantibodies remain largely elusive. We established an in vitro human astrocyte model system using induced pluripotent stem cells (iPSCs) technology in combination with NMO patient-derived serum and IgG to elucidate the cellular and functional changes caused by NMO-IgG. Herein, we observed that NMO-IgG induces structural alterations in mitochondria and their association with the endoplasmic reticulum (ER) and lysosomes at the ultrastructural level, which potentially leads to impaired mitochondrial functions and dynamics. Indeed, human astrocytes display impaired mitochondrial bioenergetics and autophagy activity in the presence of NMO-IgG. We further demonstrated NMO-IgG-driven ER membrane deformation into a multilamellar structure in human astrocytes. Together, we show that NMO-IgG rearranges cellular organelles and alter their functions and that our in vitro system using human iPSCs offers previously unavailable experimental opportunities to study the pathophysiological mechanisms of NMO in human astrocytes or conduct large-scale screening for potential therapeutic compounds targeting astrocytic abnormalities in patients with NMO.

Pathogen-induced inflammation is attenuated by the iminosugar MON-DNJ via modulation of the unfolded protein response.

Sepsis is a life-threatening condition involving a dysregulated immune response to infectious agents that cause injury to host tissues and organs. Current treatments are limited to early administration of antibiotics and supportive care. While appealing, the strategy of targeted inhibition of individual molecules in the inflammatory cascade has not proved beneficial. Non-targeted, systemic immunosuppression with steroids has shown limited efficacy and raises concern for secondary infection. Iminosugars are a class of small molecule glycomimetics with distinct inhibition profiles for glycan processing enzymes based on stereochemistry. Inhibition of host endoplasmic reticulum resident glycoprotein processing enzymes has demonstrated efficacy as a broad-spectrum antiviral strategy, but limited consideration has been given to the effects on host glycoprotein production and consequent disruption of signalling cascades. This work demonstrates that iminosugars inhibit dengue virus, bacterial lipopolysaccharide and fungal antigen-stimulated cytokine responses in human macrophages. In spite of decreased inflammatory mediator production, viral replication is suppressed in the presence of iminosugar. Transcriptome analysis reveals the key interaction of pathogen-induced endoplasmic reticulum stress, the resulting unfolded protein response and inflammation. Our work shows that iminosugars modulate these interactions. Based on these findings, we propose a new therapeutic role for iminosugars as treatment for sepsis-related inflammatory disorders associated with excess cytokine secretion.

Endoplasmic Reticulum-Mitochondria Crosstalk and Beta-Cell Destruction in Type 1 Diabetes.

Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In response to inflammatory signals, beta-cells engage adaptive mechanisms where the endoplasmic reticulum (ER) and mitochondria act in concert to restore cellular homeostasis. In the recent years it has become clear that this adaptive phase may trigger the development of autoimmunity by the generation of autoantigens recognized by autoreactive CD8 T cells. The participation of the ER stress and the unfolded protein response to the increased visibility of beta-cells to the immune system has been largely described. However, the role of the other cellular organelles, and in particular the mitochondria that are central mediator for beta-cell survival and function, remains poorly investigated. In this review we will dissect the crosstalk between the ER and mitochondria in the context of T1D, highlighting the key role played by this interaction in beta-cell dysfunctions and immune activation, especially through regulation of calcium homeostasis, oxidative stress and generation of mitochondrial-derived factors.

Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.

It has been previously reported that targeting and retaining antigens in the endoplasmic reticulum (ER) can induce an ER stress response. In this study, we evaluated the antitumor effect of E7 antigen fused to an ERresident protein, cyclooxygenase-2, which possesses a 19-aminoacid cassette that directs it to the endoplasmic reticulum-associated protein degradation (ERAD) pathway. The featured DNA constructs, COX2-E7 and COX2-E7ΔERAD, with a deletion in the 19-aminoacid cassette, were used to evaluate the importance of this sequence. In vitro analysis of protein expression and ER localisation were verified. We observed that both constructs induced an ER stress response. This finding correlated with the antitumor effect in mice injected with TC-1 cells and treated with different DNA constructs by biolistic vaccination. Immunisation with COX2-E7 and COX2-E7ΔERAD DNA constructs induced a significant antitumor effect in mice, without a significant difference between them, although the COX2-E7 construct induced a significant E7-specific immune response. These results demonstrate that targeting the E7 antigen to the ERAD pathway promotes a potent therapeutic antitumor effect. This strategy could be useful for the design of other antigen-specific therapies.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Conformational sensing of major histocompatibility complex (MHC) class I molecules by immune receptors and intracellular assembly factors.

Major histocompatibility complex class I (MHC-I) molecules play a critical role in both innate and adaptive immune responses. The heterodimeric complex of a polymorphic MHC-I heavy chain and a conserved light chain binds to a diverse set of peptides which are presented at the cell surface. Peptide-free (empty) versions of MHC-I molecules are typically retained intracellularly due to their low stability and bound by endoplasmic reticulum chaperones and assembly factors. However, emerging evidence suggests that at least some MHC-I allotypes are relatively stable and detectable at the cell-surface as peptide-deficient conformers, under some conditions. Such MHC-I conformers interact with multiple immune receptors to mediate various immunological functions. Furthermore, conformational sensing of MHC-I molecules by intracellular assembly factors and endoplasmic reticulum chaperones influences the peptide repertoire, with profound consequences for immunity. In this review, we discuss recent advances relating to MHC-I conformational variations and their pathophysiological implications.

TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming.

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.

Combined immunodeficiency due to a mutation in the γ1 subunit of the coat protein I complex.

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1K652E mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.

Common allotypes of ER aminopeptidase 1 have substrate-dependent and highly variable enzymatic properties.

Polymorphic variation of immune system proteins can drive variability of individual immune responses. Endoplasmic reticulum aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by major histocompatibility complex class I molecules. Coding SNPs in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes has not been thoroughly explored. We used phased genotype data to estimate ERAP1 allotype frequency in 2504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the ten most common ERAP1 allotypes, and systematically characterized their in vitro enzymatic properties. We find that ERAP1 allotypes possess a wide range of enzymatic activities, up to 60-fold, whose ranking is substrate dependent. Strikingly, allotype 10, previously associated with Behçet's disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a subactive ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multistep trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site suggest that allotypic variation influences the communication between the regulatory and the active site. Our work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with major histocompatibility complex haplotypes, to immune response variability and predisposition to chronic inflammatory conditions.

ER-to-Golgi transport and SEC23-dependent COPII vesicles regulate T cell alloimmunity.

T cell-mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway. To study the functional role of this pathway, we generated mice with a T cell-specific deletion in SEC23B, a core subunit of coat protein complex II (COPII). We found that SEC23B critically regulated the T cell secretome following activation. SEC23B-deficient T cells exhibited a proliferative defect and reduced effector functions in vitro, as well as in experimental models of allogeneic and xenogeneic hematopoietic cell transplantation in vivo. However, T cells derived from 3 patients with congenital dyserythropoietic anemia II (CDAII), which results from Sec23b mutation, did not exhibit a similar phenotype. Mechanistic studies demonstrated that unlike murine KO T cells, T cells from patients with CDAII harbor increased levels of the closely related paralog, SEC23A. In vivo rescue of murine KO by expression of Sec23a from the Sec23b genomic locus restored T cell functions. Together, our data demonstrate a critical role for the COPII pathway, with evidence for functional overlap in vivo between SEC23 paralogs in the regulation of T cell immunity in both mice and humans.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.

Endoplasmic Reticulum Stress-Associated Neuronal Death and Innate Immune Response in Neurological Diseases.

Neuronal death and inflammatory response are two common pathological hallmarks of acute central nervous system injury and chronic degenerative disorders, both of which are closely related to cognitive and motor dysfunction associated with various neurological diseases. Neurological diseases are highly heterogeneous; however, they share a common pathogenesis, that is, the aberrant accumulation of misfolded/unfolded proteins within the endoplasmic reticulum (ER). Fortunately, the cell has intrinsic quality control mechanisms to maintain the proteostasis network, such as chaperone-mediated folding and ER-associated degradation. However, when these control mechanisms fail, misfolded/unfolded proteins accumulate in the ER lumen and contribute to ER stress. ER stress has been implicated in nearly all neurological diseases. ER stress initiates the unfolded protein response to restore proteostasis, and if the damage is irreversible, it elicits intracellular cascades of death and inflammation. With the growing appreciation of a functional association between ER stress and neurological diseases and with the improved understanding of the multiple underlying molecular mechanisms, pharmacological and genetic targeting of ER stress are beginning to emerge as therapeutic approaches for neurological diseases.

A splice acceptor variant in HLA-DRA affects the conformation and cellular localization of the class II DR alpha-chain.

Class II human leucocyte antigen (HLA) proteins are involved in the immune response by presenting pathogen-derived peptides to CD4+ T lymphocytes. At the molecular level, they are constituted by α/ß-heterodimers on the surface of professional antigen-presenting cells. Here, we report that the acceptor variant (rs8084) in the HLA-DRA gene mediates the transcription of an alternative version of the α-chain lacking 25 amino acids in its extracellular domain. Molecular dynamics simulations suggest this isoform undergoes structural refolding which in turn affects its stability and cellular trafficking. The short HLA-DRA isoform cannot reach the cell surface, although it is still able to bind the corresponding ß-chain. Conversely, it remains entrapped within the endoplasmic reticulum where it is targeted for degradation. Furthermore, we demonstrate that the short isoform can be transported to the cell membrane via interactions with the peptide-binding site of canonical HLA heterodimers. Altogether, our findings indicate that short HLA-DRA functions as a novel intact antigen for class II HLA molecules.

Apolipoprotein H drives hepatitis B surface antigen retention and endoplasmic reticulum stress during hepatitis B virus infection.

BACKGROUND: Apolipoprotein H (APOH), also known as beta2-glycoprotein I (beta2-GPI), is an acute phase protein in hepatitis B virus (HBV) infection and binds to hepatitis B surface antigen (HBsAg) with high-affinity. APOH expression is upregulated by HBV and the large surface protein (LHBs), but also elevated in HBV-related hepatoma cells. Previous studies show that intracellular retention of HBsAg induces endoplasmic reticulum (ER) stress, a key driver of hepatocyte damage during chronic liver injury, but the mechanisms are unclear. We hypothesize that APOH mediates HBV-induced ER stress through increased retention of HBsAg. METHODS: VR-APOH-myc and VR-LHBs-flag plasmids were constructed by PCR using pcDNA3.1(-)-APOH or an HBV expression vector, respectively. APOH and ER stress markers were examined at protein and mRNA levels by Western Blot or RT-qPCR. HBsAg titer was assayed by ELISA. RNA-seq was performed to elucidate the transcriptional impact of APOH manipulation in HBV-producing cells (HepG2.2.15 cells). RESULTS: We found that HBV upregulates APOH expression in 293 T cells, and APOH overexpression subsequently inhibits secretion of HBsAg. Next, we show that LHBs overexpression in conjunction with APOH leads to ER stress in 293 T cells, as evidenced by production of the binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), as well as increased splicing of X-box binding protein 1 (XBP1). We further observed that loss of beta2-GPI reduced CHOP expression in HepG2.2.15 cells, while beta2-GPI overexpression enhanced CHOP production. CONCLUSION: The interaction of beta2-GPI and HBV initiates ER stress through driving intracellular retention of HBsAg and activates the UPR.

Endoplasmic reticulum stress signals in the tumour and its microenvironment.

Protein handling, modification and folding in the endoplasmic reticulum (ER) are tightly regulated processes that determine cell function, fate and survival. In several tumour types, diverse oncogenic, transcriptional and metabolic abnormalities cooperate to generate hostile microenvironments that disrupt ER homeostasis in malignant and stromal cells, as well as infiltrating leukocytes. These changes provoke a state of persistent ER stress that has been demonstrated to govern multiple pro-tumoural attributes in the cancer cell while dynamically reprogramming the function of innate and adaptive immune cells. Aberrant activation of ER stress sensors and their downstream signalling pathways have therefore emerged as key regulators of tumour growth and metastasis as well as response to chemotherapy, targeted therapies and immunotherapy. In this Review, we discuss the physiological inducers of ER stress in the tumour milieu, the interplay between oncogenic signalling and ER stress response pathways in the cancer cell and the profound immunomodulatory effects of sustained ER stress responses in tumours.