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.

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.

Calcium storage in multivesicular endo-lysosome.

It is now established that endo-lysosomes, also referred to as late endosomes, serve as intracellular calcium store, in addition to the endoplasmic reticulum. While abundant calcium-binding proteins provide the latter compartment with its calcium storage capacity, essentially nothing is known about the mechanism responsible for calcium storage in endo-lysosomes. In this paper, we propose that the structural organization of endo-lysosomal membranes drives the calcium storage capacity of the compartment. Indeed, endo-lysosomes exhibit a characteristic multivesicular ultrastructure, with intralumenal membranes providing a large amount of additional bilayer surface. We used a theoretical approach to investigate the calcium storage capacity of endosomes, using known calcium binding affinities for bilayers and morphological data on endo-lysosome membrane organization. Finally, we tested our predictions experimentally after Sorting Nexin 3 depletion to decrease the intralumenal membrane content. We conclude that the major negatively-charge lipids and proteins of endo-lysosomes serve as calcium-binding molecules in the acidic calcium stores of mammalian cells, while the large surface area of intralumenal membranes provide the necessary storage capacity.

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 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.

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.

Discovery and validation of new Hv1 proton channel inhibitors with onco-therapeutic potential.

The voltage-gated hydrogen channel Hv1 encoded in humans by the HVCN1 gene is a highly selective proton channel that allows large fluxes of protons across biological membranes. Hv1 form functional dimers of four transmembrane spanning proteins resembling the voltage sensing domain of potassium channels. Each subunit is highly selective for protons and is controlled by changes in the transmembrane voltage and pH gradient. Hv1 is most expressed in phagocytic cells where it sustains NADPH oxidase-dependent bactericidal function and was reported to facilitate antibody production by B cells and to promote the maturation and motility of spermatocytes. Hv1 contributes to neuroinflammation following brain damage and favors cancer progression possibly by extruding protons generated during aerobic glycolysis of cancer cells. Lack of specific Hv1 inhibitors has hampered translation of this knowledge to treat immune, fertility, or malignancy diseases. In this study, we show that the genetic deletion of Hv1 delays tumor development in a mouse model of granulocytic sarcoma and report the discovery and characterization of two novel bioavailable inhibitors of Hv1 channels that we validate by orthogonal assays and electrophysiological recordings.

TMBIM5 is the Ca2+ /H+ antiporter of mammalian mitochondria.

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

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.

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.

The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex.

The stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that regulates the activity of Orai plasma membrane Ca2+ channels to mediate the store-operated Ca2+ entry pathway essential for immunity. Uncoordinated 93 homolog B1 (UNC93B1) is a multiple membrane-spanning ER protein that acts as a trafficking chaperone by guiding nucleic-acid sensing toll-like receptors to their respective endosomal signaling compartments. We previously showed that UNC93B1 interacts with STIM1 to promote antigen cross-presentation in dendritic cells, but the STIM1 binding site(s) and activation step(s) impacted by this interaction remained unknown. In this study, we show that UNC93B1 interacts with STIM1 in the ER lumen by binding to residues in close proximity to the transmembrane domain. Cysteine crosslinking in vivo showed that UNC93B1 binding promotes the zipping of transmembrane and proximal cytosolic helices within resting STIM1 dimers, priming STIM1 for translocation. In addition, we show that UNC93B1 deficiency reduces store-operated Ca2+ entry and STIM1-Orai1 interactions and targets STIM1 to lighter ER domains, whereas UNC93B1 expression accelerates the recruitment of STIM1 to cortical ER domains. We conclude that UNC93B1 therefore acts as a trafficking chaperone by maintaining the pool of resting STIM1 proteins in a state primed for activation, enabling their rapid translocation in an extended conformation to cortical ER signaling compartments.

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.

Simultaneous determination of intraluminal lysosomal calcium and pH by dextran-conjugated fluorescent dyes.

The lysosome is the main catabolic organelle in the cell, also serving as a signaling platform. Lysosomes maintain a low intraluminal pH where dozens of hydrolytic enzymes degrade a wide variety of macromolecules. Besides degradation of polymers, the lysosome is involved in various cellular processes, including energy metabolism, plasma membrane repair and antigen presentation. Recent work has shown that the lysosome is an important calcium store, modulating diverse cellular functions such as membrane fusion and fission, autophagy and lysosomal biogenesis. Precise measurement of free lysosomal calcium concentration has been hampered by its low luminal pH, since the affinity of most calcium probes decreases with higher proton concentration. Here we detailed an adapted protocol for the simultaneous measurement of lysosomal pH and calcium using dextran-conjugated ratiometric fluorescent dyes. As compared with indirect measurements of lysosomal calcium release using genetically-encoded calcium indicators (GECIs), the present method offers the possibility of obtaining pH-corrected, intraluminal calcium concentrations at single lysosome resolution. It also enables simultaneous temporal resolution of lysosomal calcium and pH.

Proteins Interacting with STIM1 and Store-Operated Ca2+ Entry.

The endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) interacts with ORAI Ca2+ channels at the plasma membrane to regulate immune and muscle cell function. The conformational changes underlying STIM1 activation, translocation, and ORAI1 trapping and gating, are stringently regulated by post-translational modifications and accessory proteins. Here, we review the recent progress in the identification and characterization of ER and cytosolic proteins interacting with STIM1 to control its activation and deactivation during store-operated Ca2+ entry (SOCE).

Molecular Mechanisms of Calcium Signaling During Phagocytosis.

Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of numerous cellular functions including vesicular trafficking, cytoskeletal rearrangements and gene transcription. Both global as well as localized Ca2+ signals occur during phagocytosis, although their functional impact on the phagocytic process has been debated. After nearly 40 years of research, a consensus may now be reached that although not strictly required, Ca2+ signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions. In the last decade many efforts have been made to identify the channels and regulators involved in generating and shaping phagocytic Ca2+ signals. While molecules involved in store-operated calcium entry (SOCE) of the STIM and ORAI family have taken center stage, members of the canonical, melastatin, mucolipin and vanilloid transient receptor potential (TRP), as well as purinergic P2X receptor families are now recognized to play significant roles. In this chapter, we review the recent literature on research that has linked specific Ca2+-permeable channels and regulators to phagocytic function. We highlight the fact that lipid mediators are emerging as important regulators of channel gating and that phagosomal ionic homeostasis and Ca2+ release also play essential parts. We predict that improved methodologies for measuring these factors will be critical for future advances in dissecting the intricate biology of this fascinating immune process.

Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein.

Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2 O2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays.

Ultrastructural Characterization of Flashing Mitochondria.

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ~1,200 slices of 10 nm thickness from a 28 µm × 15 µm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.