Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.

SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis.

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated on endoplasmic reticulum (ER) cisternae juxtaposed to phagocytic cups when expressed in phagocytic COS-7 cells. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE) and receptor-mediated phagocytosis, stalling particle capture at the cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2 and Nir3 (Nir2/3) double-knockout cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 depletion reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir proteins maintain phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis.

Enforced tethering elongates the cortical endoplasmic reticulum and limits store-operated Ca2+ entry.

Recruitment of STIM proteins to cortical endoplasmic reticulum (cER) domains forming membrane contact sites (MCSs) mediate the store-operated Ca2+ entry (SOCE) pathway essential for human immunity. The cER is dynamically regulated by STIM and tethering proteins during SOCE, but the ultrastructural rearrangement and functional consequences of cER remodeling are unknown. Here, we express natural (E-Syt1 and E-Syt2) and artificial (MAPPER-S and MAPPER-L) protein tethers in HEK-293T cells and correlate the changes in cER length and gap distance, as measured by electron microscopy, with ionic fluxes. We found that native cER cisternae extended during store depletion and remained elongated at a constant ER-plasma membrane (PM) gap distance during subsequent Ca2+ elevations. Tethering proteins enhanced store-dependent cER expansion, anchoring the enlarged cER at tether-specific gap distances of 12-15 nm (E-Syts) and 5-9 nm (MAPPERs). Cells with artificially extended cER had reduced SOCE and reduced agonist-induced Ca2+ release. SOCE remained modulated by calmodulin and exhibited enhanced Ca2+-dependent inhibition. We propose that cER expansion mediated by ER-PM tethering at a close distance negatively regulates SOCE by confining STIM-ORAI complexes to the periphery of enlarged cER sheets, a process that might participate in the termination of store-operated Ca2+ entry.

S-acylation of Ca2+ transport proteins: molecular basis and functional consequences.

Calcium (Ca2+) regulates a multitude of cellular processes during fertilization and throughout adult life by acting as an intracellular messenger to control effector functions in excitable and non-excitable cells. Changes in intracellular Ca2+ levels are driven by the co-ordinated action of Ca2+ channels, pumps, and exchangers, and the resulting signals are shaped and decoded by Ca2+-binding proteins to drive rapid and long-term cellular processes ranging from neurotransmission and cardiac contraction to gene transcription and cell death. S-acylation, a lipid post-translational modification, is emerging as a critical regulator of several important Ca2+-handling proteins. S-acylation is a reversible and dynamic process involving the attachment of long-chain fatty acids (most commonly palmitate) to cysteine residues of target proteins by a family of 23 proteins acyltransferases (zDHHC, or PATs). S-acylation modifies the conformation of proteins and their interactions with membrane lipids, thereby impacting intra- and intermolecular interactions, protein stability, and subcellular localization. Disruptions of S-acylation can alter Ca2+ signalling and have been implicated in the development of pathologies such as heart disease, neurodegenerative disorders, and cancer. Here, we review the recent literature on the S-acylation of Ca2+ transport proteins of organelles and of the plasma membrane and highlight the molecular basis and functional consequence of their S-acylation as well as the therapeutic potential of targeting this regulation for diseases caused by alterations in cellular Ca2+ fluxes.

RNA metabolism: putting the brake on the UPR.

The unfolded protein response (UPR) is a major signaling cascade that determines cell fate under conditions of endoplasmic reticulum (ER) stress. The kinetics and amplitude of UPR responses are tightly controlled by several feedback loops and the expression of positive and negative regulators. In this issue of EMBO Reports, the Wilkinson lab uncovers a novel function of nonsense-mediated RNA decay (NMD) in fine-tuning the UPR. NMD is an mRNA quality control mechanism known to destabilize aberrant mRNAs that contain premature termination codons. In this work, NMD was shown to determine the threshold of stress necessary to activate the UPR, in addition to adjusting the amplitude of downstream responses and the termination phase. These effects were mapped to the control of the mRNA stability of IRE1, a major ER stress transducer. This study highlights the dynamic crosstalk between mRNA metabolism and the proteostasis network demonstrating the physiological relevance of normal mRNA regulation by the NMD pathway.

Coordinated regulation of the orosomucoid-like gene family expression controls de novo ceramide synthesis in mammalian cells.

The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.

ORMDL3 modulates store-operated calcium entry and lymphocyte activation.

T lymphocytes rely on a Ca(2+) signal known as store-operated calcium entry (SOCE) for their activation. This Ca(2+) signal is generated by activation of a T-cell receptor, depletion of endoplasmic reticulum (ER) Ca(2+) stores and activation of Ca(2+) release-activated Ca(2+) currents (I(CRAC)). Here, we report that the ER protein orosomucoid like 3 (ORMDL3), the product of the ORMDL3 gene associated with several autoimmune and/or inflammatory diseases, negatively modulates I(CRAC), SOCE, nuclear factor of activated T cells nuclear translocation and interleukin-2 production. ORMDL3 inhibits the Ca(2+) influx mechanism at the outer mitochondrial membrane, resulting in a Ca(2+)-dependent inhibition of I(CRAC) and reduced SOCE. The effect of ORMDL3 could be mimicked by interventions that decreased mitochondrial Ca(2+) influx and reverted by buffering of cytosolic Ca(2+) or activation of mitochondrial Ca(2+) influx. In conclusion, ORMDL3 modifies key steps in the process of T-lymphocyte activation, providing a functional link between the genetic associations of the ORMDL3 gene with autoimmune and/or inflammatory diseases.

Extending the Contacts Breaks the Flow.

In this news and views, we discuss our recent publication where we described how ER-PM membrane contact sites (MCS) are modulated during store operated calcium entry (SOCE). We also examine why enforcing ER-PM MCS by tethering proteins does not not enhance, but rather inhibits SOCE.

The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation.

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule (STIM)-gated ORAI channels at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites maintains adequate levels of Ca2+ within the ER lumen during Ca2+ signaling. Disruption of ER Ca2+ homeostasis activates the unfolded protein response (UPR) to restore proteostasis. Here, we report that the UPR transducer inositol-requiring enzyme 1 (IRE1) interacts with STIM1, promotes ER-PM contact sites, and enhances SOCE. IRE1 deficiency reduces T cell activation and human myoblast differentiation. In turn, STIM1 deficiency reduces IRE1 signaling after store depletion. Using a CaMPARI2-based Ca2+ genome-wide screen, we identify CAMKG2 and slc105a as SOCE enhancers during ER stress. Our findings unveil a direct crosstalk between SOCE and UPR via IRE1, acting as key regulator of ER Ca2+ and proteostasis in T cells and muscles. Under ER stress, this IRE1-STIM1 axis boosts SOCE to preserve immune cell functions, a pathway that could be targeted for cancer immunotherapy.

Ion channels in asthma.

Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca(2+) levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.

Balancing energy and protein homeostasis at ER-mitochondria contact sites.

The endoplasmic reticulum (ER) is the largest organelle of the cell and participates in multiple essential functions, including the production of secretory proteins, lipid synthesis, and calcium storage. Sustaining proteostasis requires an intimate coupling with energy production. Mitochondrial respiration evolved to be functionally connected to ER physiology through a physical interface between both organelles known as mitochondria-associated membranes. This quasi-synaptic structure acts as a signaling hub that tunes the function of both organelles in a bidirectional manner and controls proteostasis, cell death pathways, and mitochondrial bioenergetics. Here, we discuss the main signaling mechanisms governing interorganellar communication and their putative role in diseases including cancer and neurodegeneration.

The TAM-associated STIM1I484R mutation increases ORAI1 channel function due to a reduced STIM1 inactivation break and an absence of microtubule trapping.

Tubular aggregate myopathy (TAM) is a progressive skeletal muscle disease associated with gain-of-function mutations in the ER Ca2+ sensor STIM1 that mediates store-operated Ca2+ entry (SOCE) across the Ca2+-release-activated (CRAC) Ca2+ channel ORAI1. A frameshift mutation in STIM1 inactivation domain, STIM1I484R, was identified in a TAM patient and reported to decrease SOCE. Using ion imaging and electrophysiology, we show that the STIM1I484R mutation instead renders STIM1 constitutively active. In ion imaging experiments, STIM1I484R was less efficient than native STIM1 when expressed alone but enhanced SOCE and increased basal Ca2+ and Mn2+ influx when expressed together with ORAI1. In patch-clamp recordings, STIM1I484R generated larger pre-activated CRAC currents lacking slow Ca2+-dependent inhibition (SCDI). STIM1I484R was pre-recruited in plasma membrane clusters when co-expressed with ORAI1, as were mutants truncated at the frameshift residue or lacking EB-1-binding, which recapitulated STIM1I484R gain-of-function. When expressed alone in human primary myoblasts, STIM1I484R was pre-recruited in large clusters and increased basal Ca2+ entry. These observations establish that STIM1I484R confers a gain of CRAC channel function due to the loss of critical inhibitory C-terminal domains that prevent STIM1 binding to ORAI1, enable STIM1 trapping by microtubules, and mediate SCDI, providing a mechanistic explanation for the muscular defects of TAM patients bearing this mutation.

S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse.

Efficient immune responses require Ca2+ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 promotes TCR recruitment and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca2+ entry in HEK-293 cells and nearly abrogated long-lasting Ca2+ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel remained in cholesterol-poor domains upon enforced ZDHHC20 expression and was recruited less efficiently to the IS along with actin and TCR. Our results establish S-acylation as a critical regulator of ORAI1 channel trafficking and function at the IS and reveal that ORAI1 S-acylation enhances TCR recruitment to the synapse.

Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1.

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics.

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.

Publisher Correction: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Calcium signaling at the endoplasmic reticulum: fine-tuning stress responses.

Endoplasmic reticulum (ER) calcium signaling is implicated in a myriad of coordinated cellular processes. The ER calcium content is tightly regulated as it allows a favorable environment for protein folding, in addition to operate as a major reservoir for fast and specific release of calcium. Altered ER homeostasis impacts protein folding, activating the unfolded protein response (UPR) as a rescue mechanism to restore proteostasis. ER calcium release impacts mitochondrial metabolism and also fine-tunes the threshold to undergo apoptosis under chronic stress. The global coordination between UPR signaling and energetic demands takes place at mitochondrial associated membranes (MAMs), specialized subdomains mediating interorganelle communication. Here we discuss current models explaining the functional relationship between ER homeostasis and various cellular responses to coordinate proteostasis and metabolic maintenance.

IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a signalling network known as the unfolded protein response (UPR). Here, we identified filamin A as a major binding partner of the ER stress transducer IRE1α. Filamin A is an actin crosslinking factor involved in cytoskeleton remodelling. We show that IRE1α controls actin cytoskeleton dynamics and affects cell migration upstream of filamin A. The regulation of cytoskeleton dynamics by IRE1α is independent of its canonical role as a UPR mediator, serving instead as a scaffold that recruits and regulates filamin A. Targeting IRE1α expression in mice affected normal brain development, generating a phenotype resembling periventricular heterotopia, a disease linked to the loss of function of filamin A. IRE1α also modulated cell movement and cytoskeleton dynamics in fly and zebrafish models. This study unveils an unanticipated biological function of IRE1α in cell migration, whereby filamin A operates as an interphase between the UPR and the actin cytoskeleton.