ER Ca2 Research Articles

Regulation of the Ca2+‐sensing capacity of STIM1 by the G protein‐coupled estrogen receptor

The stromal interaction molecule 1 (STIM1) is essential for activation of store‐operated Ca2+entry (SOCE). STIM1 contains in its intra‐endoplasmic reticular (ER) domain a canonical EF hand (cEF) that houses a Ca2+‐binding loop. This loop serves as a Ca2+sensor; Ca2+leaving its binding loop upon ER Ca2+depletion initiates conformational changes leading to STIM1 oligomerization and SOCE activation. We recently reported that activation of the G protein‐coupled estrogen receptor (GPER) clamps intracellular Ca2+signals via multifaceted inhibitory actions on both Ca2+efflux and SOCE; the latter results partly from alteration in phosphorylation at serines 575, 608 and 621 of STIM1. Here, we identified a dynamic interaction between GPER and STIM1 that directly controls the Ca2+‐sensing capacity of STIM1. GPER coimmunoprecipitates with STIM1 in resting primary endothelial cells, an association enhanced by treatment with either GPER agonist G‐1 or activation of SOCE using thapsigargin. This effect is not altered by combined S575/608/621A substitutions. Coimmunoprecipitation analysis using truncated versions of STIM1 showed that GPER associates with the cEF domain. Confocal microscopy identified a GPER's submembrane domain that localizes GPER to the ER. To assess the role of GPER‐STIM1 interaction in controlling STIM1's Ca2+sensing capacity, we generated a series of biosensors that incorporated STIM1's Ca2+‐binding loop in singlet, doublet or triplet. These biosensors faithfully report Ca2+binding to the loop, with tripled dynamic range by the triple‐loop biosensor and loss of Ca2+binding by charge substitutions targeting strategic elements for Ca2+binding. Using peptides corresponding to submembrane domains of GPER and these STIM1 biosensors, we identified a robust Ca2+‐independent interaction between a submembrane domain and the Ca2+‐binding loop. The charge substitutions that abolish Ca2+binding only reduce but do not negate the interaction, indicating that Ca2+and the GPER domain bind to different regions of the loop. Consistently, we have observed that GPER overexpression decreases SOCE and GPER knockdown substantially increases SOCE. These data reveal a new interaction between GPER and STIM1 that directly controls STIM1's Ca2+‐sensing capacity and SOCE activation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PSEN2 (presenilin 2) mutants linked to familial Alzheimer disease impair autophagy by altering Ca2+ homeostasis

ABSTRACTPSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aβ peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca2+ homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca2+ content, thus reducing cytosolic Ca2+ response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca2+ signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration.Abbreviations: Aβ: amyloid β; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A1; BAPTA-AM: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; CFP: cyan fluorescent protein; EGTA-AM: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; ER: endoplasmic reticulum; EGFP-HDQ74: enhanced GFP-huntingtin exon 1 containing 74 polyglutamine repeats; FAD: familial Alzheimer disease; FCS: fetal calf serum; FRET: fluorescence/Förster resonance energy transfer; GFP: green fluorescent protein; IP3: inositol trisphosphate; KD: knockdown; LAMP1: lysosomal associated membrane protein 1; MAP1LC3-II/LC3-II: lipidated microtubule-associated protein 1 light chain 3; MCU: mitochondrial calcium uniporter; MICU1: mitochondrial calcium uptake 1; MEFs: mouse embryonic fibroblasts; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; SQSTM1/p62: sequestosome 1; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB7: RAB7A: member RAS oncogene family; RFP: red fluorescent protein; ATP2A/SERCA: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H+-ATPase; WT: wild type

Identification of Novel RyR1 Inhibitors by High-Throughput Screening using ER Ca2+ Measurement

Identification of Novel RyR1 Inhibitors by High-Throughput Screening using ER Ca2+ Measurement

GPN does not release lysosomal Ca2+ but evokes Ca2+ release from the ER by increasing the cytosolic pH independently of cathepsin C.

ABSTRACTThe dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.

Structure development of oxolinic acid, a novel inhibitor of type 1 ryanodine receptor

Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel on the sarcoplasmic reticulum in the skeletal muscle. Mutations in RyR1 cause various muscle diseases including malignant hyperthermia (MH) and central core disease (CCD). Although dantrolene is the only therapeutic drugs for MH, it cannot be used for CCD due to its lower solubility and side effects. It is therefore urgent to discover novel RyR1 inhibitors. We have recently identified oxolinic acid as a novel RyR1 inhibitor. However, affinity of oxolinic acid was much lower than that of dantrolene. In this study, we designed and synthesized a series of quinolone derivatives using oxolinic acid as a lead compound. Dose-dependent inhibitory effects were evaluated by ER Ca2+ measurement using HEK293 cells expressing R-CEPIA1er, a genetically-encoded ER Ca2+ indicator, and RyR1 carrying an MH mutation (R2163C). Compounds bearing a longer alkyl chain at the nitrogen atom of quinolone ring exhibited stronger RyR1 inhibiting activity. Modification at 6 and 7 positions of quinolone ring greatly affected the inhibitory activity. Derivatives of oxolinic acid may be good candidates for treatment of RyR1-related diseases.

Neurite Collapse and Altered ER Ca2+ Control in Human Parkinson Disease Patient iPSC-Derived Neurons with LRRK2 G2019S Mutation.

SummaryThe Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.

Human cytomegalovirus US21 protein is a viroporin that modulates calcium homeostasis and protects cells against apoptosis

The human cytomegalovirus (HCMV) US12 gene family comprises a set of 10 contiguous genes (US12 to US21) with emerging roles in the regulation of virus cell tropism, virion composition, and immunoevasion. Of all of the US12 gene products, pUS21 shows the highest level of identity with two cellular transmembrane BAX inhibitor motif-containing (TMBIM) proteins: Bax inhibitor-1 and Golgi anti-apoptotic protein, both of which are involved in the regulation of cellular Ca2+ homeostasis and adaptive cell responses to stress conditions. Here, we report the US21 protein to be a viral-encoded ion channel that regulates intracellular Ca2+ homeostasis and protects cells against apoptosis. Indeed, we show pUS21 to be a 7TMD protein expressed with late kinetics that accumulates in ER-derived vesicles. Deletion or inactivation of the US21 gene resulted in reduced HCMV growth, even in fibroblasts, due to reduced gene expression. Ratiometric fluorescence imaging assays revealed that expression of pUS21 reduces the Ca2+ content of intracellular ER stores. An increase in cell resistance to intrinsic apoptosis was then observed as an important cytobiological consequence of the pUS21-mediated alteration of intracellular Ca2+ homeostasis. Moreover, a single point mutation in the putative pore of pUS21 impaired the reduction of ER Ca2+ concentration and attenuated the antiapoptotic activity of pUS21wt, supporting a functional link with its ability to manipulate Ca2+ homeostasis. Together, these results suggest pUS21 of HCMV constitutes a TMBIM-derived viroporin that may contribute to HCMV's overall strategy to counteract apoptosis in infected cells.

A secretory pathway kinase regulates sarcoplasmic reticulum Ca2+ homeostasis and protects against heart failure.

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

Calcium sensing by the STIM1 ER-luminal domain

Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5–6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.

Role of calcium in adult onset polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in genes encoding the polycystin (PC) 1 and 2 proteins. The goal of this study was to determine the role of calcium in regulating cyst growth. Stromal interaction molecule 1 (STIM1) protein expression was 15-fold higher in PC1-null proximal tubule cells (PN) than in heterozygote (PH) controls and 2-fold higher in an inducible, PC1 knockout, mouse model of ADPKD compared to a non-cystic match control. IP3 receptor protein expression was also higher in the cystic mice. Knocking down STIM1 with siRNA reduced cyst growth and lowered cAMP levels in PN cells. Fura2 measurements of intracellular Ca2+ showed higher levels of intracellular Ca2+, SOCE and thaspigargin-stimulated ER Ca2+ release in PN vs. PH cells. There was a dramatic reduction in thapsigargin-stimulated release of ER Ca2+ following STIM1 silencing or application of 2-APB, consistent with altered ER Ca2+ movement; the protein expression of the Ca2+-dependent adenylyl cyclases (AC) AC3 and AC6 was up- and down-regulated, respectively. Like STIM1 knockdown, application of the calmodulin inhibitor W7 lowered cAMP levels, further indicating that STIM1 regulates AC3 via Ca2+ We conclude that the high levels of STIM1 in ADPKD cells play a role in supporting cyst growth and promoting high cAMP levels and an increased release of Ca2+ from the ER. Thus, our results provide novel therapeutic targets for treating ADPKD.

Abstract 126: Nox5 Regulation of Vascular Contraction Involves Oxidation of Endoplasmic Reticulum Calcium Channels and Calreticulin

The biological function of calcium-sensitive superoxide-generating Nox5 is unclear, but it may play a role in regulating contraction, as we previously demonstrated. Here we explored molecular mechanisms whereby Nox5 controls contraction. Human arteries and mice expressing human NOX5 in smooth muscle cells (Nox5+SM22+) were studied. In arteries from hypertensive subjects, Nox5 expression, assessed by immunoblotting, was increased (50%, p<0.05 vs control). In human VSMCs, AngII-induced ROS generation (1 fold) and activation of myosin light chain (MLC) (2.5 fold) were exaggerated in VSMCs from hypertensive subjects (p<0.05 vs control); an effect that was attenuated by Nox5 siRNA. In arteries from Nox5+/SM22+ mice, contraction to U46619 was increased in (5.8±0.3 mN vs WT: 4.2±0.2 mN, p<0.05). These hypercontractile responses were inhibited by NAC (ROS scavenger), calmidazolium (calmodulin inhibitor), dantrolene (ryanodine receptor Ca 2+ channel inhibitors) and CDN1163 (SERCA channel activator), but not by a Nox1/Nox4 inhibitor (GKT137831). ONOO - levels were increased in vessels from Nox5+/SM22+ mice (5.8±0.9 vs WT 3.4±0.1 AU/mg, p<0.05). Inactivation of MYPT1 (181±1.8AU vs 164±1.9AU WT) and activation of MLC (207±10.3AU vs 155±2.7AU WT) were increased in VSMCs from Nox5+SM22+ (p<0.05). To assess the oxidative proteome in VSMCs, we immunoprecipitated reversibly oxidized proteins and observed oxidation of Nox5, decreased oxidation of MYPT1 and increased oxidation of SERCA2b in Nox5+/SM22+ mice . Proteome analysis of human VSMCs identified the ER Ca 2+ sensor, calreticulin, as a potential Nox5 binding protein. Calreticulin reversible oxidation was increased in VSMCs from Nox5+SM22+ mice and hypertensive subjects. Our study unravels crosstalk between oxidative stress and Ca 2+ in the vasculature, where Nox5 regulation of contraction involves ROS, Ca 2+ and endoplasmic reticulum localized Ca 2+ channels/proteins. We identify novel mechanisms whereby Nox5 influences pro-contractile signalling through processes involving oxidation of the ER Ca 2+ sensor, calreticulin, and ER Ca 2+ channels.

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (Aβ) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca2+ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4RΔC20), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8–10 nm) and the long- (40–50 nm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca2+ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca2+ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4RΔC20 tau, indicating that Ca2+ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies.

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11–491 and STIM11–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

Plasmodium falciparum histidine triad protein and calmodulin modulates calcium homeostasis and intracellular proteolysis

Calcium signaling has an essential role in fundamental processes of Plasmodium life cycle, including migration, cell invasion and parasite development. Two important players in calcium homeostasis, the Histidine Triad (HIT) protein that is implicated in calcium signaling in mammalian cells and calmodulin, which is a classic calcium sensor in eukaryotes are present in Plasmodium falciparum, however theirs function is unknown in the parasite. Here, we investigated the involvement of the P. falciparum Histidine Triad protein (PfHint-1) and calmodulin (PfCaM) in calcium signaling and intracellular proteolysis. For this, we targeted PfHint-1 with a hemagglutinin tail and overexpressed both proteins. We observed that PfHint-1 is expressed throughout the erythrocytic stages and partially colocalizes to the endoplasmic reticulum. Parasites overexpressing PfHint-1 displayed lower ER Ca2+ content and a higher [Ca2+]cyt rise in the parasite cytosol upon Ca2+ addition to the extracellular medium after depletion of ER calcium store. PfCaM-overexpressing parasites exhibit a higher [Ca2+]cyt rise after challenge with the calmodulin inhibitor, calmidazolium. The calcium-dependent proteolytic activity in PfCaM- and PfHint-1-overexpressing parasites was increased and correlated to alterations in calcium homeostasis. Taken together, our results indicate the participation of these proteins in P. falciparum fundamental cellular processes and highlights promising targets for the development of antimalarial drugs.

STIM1 Knockout Enhances PDGF-Mediated Ca2+ Signaling through Upregulation of the PDGFR⁻PLCγ⁻STIM2 Cascade.

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca2+ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR–PLCγ–STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ–IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.

SOCE mediated by STIM and Orai is essential for pacemaker activity in the interstitial cells of Cajal in the gastrointestinal tract.

Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca2+ from intracellular stores that stimulates Ca2+-activated Cl- channels (CaCCs) in the plasma membrane. Thus, Ca2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca2+ entry (SOCE) facilitates the refilling of Ca2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca2+ release-activated current (ICRAC). An inhibitory peptide that interfered with the STIM-Orai interaction blocked ICRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca2+ transients in ICC. These data suggest that Ca2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity.

A charge-sensing region in the stromal interaction molecule 1 luminal domain confers stabilization-mediated inhibition of SOCE in response to S-nitrosylation

Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal interaction molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ depletion-dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.

Nogo-C regulates post myocardial infarction fibrosis through the interaction with ER Ca2+ leakage channel Sec61\u03b1 in mouse hearts

Cardiac fibrosis is an independent risk factor for heart failure and even the leading cause of death in myocardial infarction patients. However, molecular mechanisms associated with the pathogenesis of cardiac fibrosis following myocardial infarction are not yet fully understood. Nogo-C protein ubiquitously expresses in tissues including in the heart. Our previous study found that Nogo-C regulated cardiomyocyte apoptosis during myocardial infarction. In the present study, we found that Nogo-C was upregulated in fibrotic hearts after myocardial infarction and in Ang II- or TGF-β1-stimulated cardiac fibroblasts. Overexpression of Nogo-C in cardiac fibroblasts increased expression of pro-fibrogenic proteins, while knockdown of Nogo-C inhibited the fibrotic responses of cardiac fibroblasts to Ang II- or TGF-β1 stimulation. Functionally, Nogo-C deficiency suppressed pro-fibrogenic proteins in post-myocardial infarction hearts and ameliorated post-myocardial infarction cardiac function. Mechanistically, we found that Nogo-C increased intracellular Ca2+ concentration and buffering Ca2+ totally abolished Nogo-C-induced fibrotic responses. Moreover, overexpression of Nogo-C caused increased Sec61α, the Ca2+ leakage channel on endoplasmic reticulum membrane. Nogo-C interacted with Sec61α on endoplasmic reticulum and stabilized Sec61α protein by inhibiting its ubiquitination. Inhibition or knockdown of Sec61α blocked Nogo-C-induced increase of cytosolic Ca2+ concentration and inhibited Nogo-C- and TGF-β1-induced fibrotic responses in cardiac fibroblasts, suggesting that Nogo-C regulates cardiac fibrosis through interacting with Sec61α to mediate the Ca2+ leakage from endoplasmic reticulum. Thus, our results reveal a novel mechanism underlying cardiac fibrosis following myocardial infarction, and provide a therapeutic strategy for cardiac remodeling related heart diseases.

The ER Ca2+ sensor STIM1 can activate osteoblast and odontoblast differentiation in mineralized tissues

ABSTRACTBone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca2+ stores and this store depletion is sensed by the ER Ca2+ sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.

Facilitation of hippocampal long-term potentiation and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking learning, memory, and the potential eradication of HIV-1

Learning and memory are generally considered the behavioral correlates of long-term potentiation (LTP), a form of synaptic plasticity associated with a persistent and long-lasting increase in synaptic strength. Repetitive stimulation of excitatory synapses in the hippocampal CA1 region leads to release and binding of glutamate to the glutamate receptors AMPAR and NMDAR located on pyramidal neurons. Activation of AMPARs facilitates Na+ influx, postsynaptic depolarization, NMDAR-mediated Ca2+ influx, and activation of several intracellular mechanisms that characterize LTP, including increased AMPAR synthesis, ROS production, and ER Ca2+ release. BDNF-TrkB receptor signaling, which increases intracellular Ca2+ levels via PLCγ1-mediated ER Ca2+ release, also plays an important role in facilitating hippocampal CA1 LTP. Interestingly, the cellular mechanisms that characterize LTP are strikingly similar to signaling pathways that underlie reactivation of latent HIV-1 reservoirs. Known as the “shock and kill” approach, reactivation of latent HIV-1, particularly in CD4+ memory T cells, is currently being pursued to potentially eradicate HIV-1. Indeed, AMPARs, NMDARs, and TrkB receptors have been found on and promote T cell activation and BDNF has been shown to reactivate latent HIV-1 in human macrophages. Additionally, latent HIV-1 reactivation via T cell receptor activation (a positive control in HIV-1 latency studies) involves PLCγ1-mediated increases in intracellular Ca2+, an increase in ROS levels, and activation of kinases and transcription factors that are also critical for LTP. Furthermore, PMA, also used as a positive control along with ionomycin in HIV-1 latency studies, has been shown to enhance hippocampal CA1 LTP. AMPK, an evolutionarily conserved kinase activated by increases in intracellular Ca2+, ROS, and/or AMP/ATP ratio increases improves lifespan and healthspan in several model organisms and is essential for T cell activation. Knockdown of AMPK also significantly inhibits HIV-1 replication. AMPK has been found localized in hippocampal CA1 dendrites and glutamate, NMDA, KCl, ionomycin, and BDNF have each been shown to induce AMPK activation in neurons. AMPK activation also increases synthesis and membrane insertion of AMPARs. Because both T cell activation and LTP are dependent on intracellular Ca2+ increases and because inhibition of ROS significantly inhibits hippocampal CA1 LTP and T cell activation, it is our hypothesis that AMPK links latent HIV-1 reactivation with hippocampal LTP, learning, and memory. We also propose that compounds that enhance or promote LTP and reactivate latent HIV-1 (e.g. PMA, ionomycin, resveratrol, metformin, etc.) either alone or in combination likely do so via AMPK activation.