Sarco/endoplasmic Reticulum Ca2+-ATPase Research Articles

The Functioning of Na+-ATPases from Protozoan Parasites: Are These Pumps Targets for Antiparasitic Drugs?

The ENA ATPases (from exitus natru: the exit of sodium) belonging to the P-type ATPases are structurally very similar to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA); they exchange Na+ for H+ and, therefore, are also known as Na+-ATPases. ENA ATPases are required in alkaline milieu, as in the case for Aspergillus, where other transporters cannot mediate an uphill Na+ efflux. They are also important for salt tolerance, as described for Arabidopsis. During their life cycles, protozoan parasites might encounter a high pH environment, thus allowing consideration of ENA ATPases as possible targets for controlling certain severe parasitic diseases, such as Chagas’ Disease. Phylogenetic analysis has now shown that, besides the types IIA, IIB, IIC, and IID P-type ATPases, there exists a 5th subgroup of ATPases classified as ATP4-type ATPases, found in Plasmodium falciparum and Toxoplasma gondii. In malaria, for example, some drugs targeting PfATP4 destroy Na+ homeostasis; these drugs, which include spiroindolones, are now in clinical trials. The ENA P-type (IID P-type ATPase) and ATP4-type ATPases have no structural homologue in mammalian cells, appearing only in fungi, plants, and protozoan parasites, e.g., Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium falciparum. This exclusivity makes Na+-ATPase a potential candidate for the biologically-based design of new therapeutic interventions; for this reason, Na+-ATPases deserves more attention.

Modulation of SERCA2a expression and function by ultrasound-guided myocardial gene transfection.

Sarco/endoplasmic reticulum Ca²+-ATPase (SERCA2a) is important for cardiac physiological function and pathological progression. However, intravenous injection, a commonly applied approach for gene delivery in most studies investigating the expression of SERCA2a in cardiomyocytes, has not been particularly satisfactory. Therefore, in the present study, a modified method was used to transfect this gene into the heart. Specifically, a SERCA2a-knockdown lentivirus was directly injected into the myocardium of adult rats under ultrasound guidance, following which the effectiveness and feasibility of this proposed approach were evaluated. The results demonstrated that compared with traditional intravenous injection, the modified gene delivery method resulted in markedly higher transfection efficiency. In addition, the SERCA2a-knockdown rats exhibited higher rates of arrhythmia and weaker ventricular wall motions compared with those in the control rats, with these symptoms more evident in the rats that received a direct injection into the myocardium compared with those that were intravenously injected. These results suggest that ultrasound-guided injection into the myocardium is an efficient and safe method for gene delivery and for inducing the knockdown of SERCA2a protein expression in cardiomyocytes in their native environment.

TRPV4 contributes to ER stress: Relation to apoptosis in the MPP+-induced cell model of Parkinson's disease

AimsParkinson's disease (PD) is a multifactorial neurodegenerative disorder. Its molecular mechanism is still unclear. Endoplasmic reticulum (ER) stress has been highlighted in PD. Transient receptor potential vanilloid 4 (TRPV4) is a kind of nonselective calcium cation channel. A defined role for TRPV4 in PD has not been reported. The purpose of the present research was to investigate the molecular mechanisms by which TRPV4 regulates ER stress induced by the 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. Main methodsPC12 cells were pretreated with the TRPV4-specific antagonist HC067047 or transfected with TRPV4 siRNA followed by treatment with MPP+. Cell viability was measured by the CCK-8 Assay. The expression of TRPV4, sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), C/EBP homologous protein (CHOP), procaspase-12, and tyrosine hydroxylase (TH) was detected by western blot and RT-PCR. Key findingsThe expression of TRPV4 was upregulated, while cell viability was decreased by MPP+, which was reversed by HC067047. The ER stress common molecular signature SERCA2 was depressed by MPP+. Moreover, MPP+ induced upregulation of GRP78, GRP94, CHOP, and decrease in procaspase-12 and TH. HC067047 and TRPV4 siRNA reversed MPP+-induced ER stress and restored TH production. SignificanceTRPV4 functions upstream of ER stress induced by MPP+ and holds promise as a prospective pharmacotherapy target for PD.

Metabolic syndrome inhibits store-operated Ca2+ entry and calcium-induced calcium-release mechanism in coronary artery smooth muscle

Background and purposeMetabolic syndrome causes adverse effects on the coronary circulation including altered vascular responsiveness and the progression of coronary artery disease (CAD). However the underlying mechanisms linking obesity with CAD are intricated. Augmented vasoconstriction, mainly due to impaired Ca2+ homeostasis in coronary vascular smooth muscle (VSM), is a critical factor for CAD. Increased calcium–induced calcium release (CICR) mechanism has been associated to pathophysiological conditions presenting persistent vasoconstriction while increased store operated calcium (SOC) entry appears to activate proliferation and migration in coronary vascular smooth muscle (VSM). We analyze here whether metabolic syndrome might alter SOC entry as well as CICR mechanism in coronary arteries, contributing thus to a defective Ca2+ handling and therefore accelerating the progression of CAD. Experimental approachMeasurements of intracellular Ca2+ ([Ca2+]i) and tension and of Ca2+ channels protein expression were performed in coronary arteries (CA) from lean Zucker rats (LZR) and obese Zucker rats (OZR). Key resultsSOC entry stimulated by emptying sarcoplasmic reticulum (SR) Ca2+ store with cyclopiazonic acid (CPA) was decreased and associated to decreased STIM-1 and Orai1 protein expression in OZR CA. Further, CICR mechanism was blunted in these arteries but Ca2+ entry through voltage-dependent L-type channels was preserved contributing to maintain depolarization-induced increases in [Ca2+]i and vasoconstriction in OZR CA. These results were associated to increased expression of voltage-operated L-type Ca2+ channel alpha 1C subunit (CaV1.2) but unaltered ryanodine receptor (RyR) and sarcoendoplasmic reticulum Ca2+‐ATPase (SERCA) pump protein content in OZR CA. Conclusion and implicationsThe present manuscript provides evidence of impaired Ca2+ handling mechanisms in coronary arteries in metabolic syndrome where a decrease in both SOC entry and CICR mechanism but preserved vasoconstriction are reported in coronary arteries from obese Zucker rats. Remarkably, OZR CA VSM at this state of metabolic syndrome seemed to have developed a compensation mechanism for impaired CICR by overexpressing CaV1.2 channels.

Neuronatin regulates whole-body metabolism: is thermogenesis involved?

Neuronatin (NNAT) was originally discovered in 1995 and labeled as a brain developmental gene due to its abundant expression in developing brains. Over the past 25 years, researchers have uncovered NNAT in other tissues; notably, the hypothalamus, pancreatic β‐cells, and adipocytes. Recent evidence in these tissues indicates that NNAT plays a significant role in metabolism whereby it regulates food intake, insulin secretion, and adipocyte differentiation. Furthermore, genetic deletion of Nnat in mice lowers whole‐body energy expenditure and increases susceptibility to diet‐induced obesity and glucose intolerance; however, the underlying cellular mechanisms remain unknown. Based on its sequence homology with phospholamban, NNAT has a purported role in regulating the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump. However, NNAT also shares sequence homology with sarcolipin, which has the unique property of uncoupling the SERCA pump, increasing whole‐body energy expenditure and thus promoting adaptive thermogenesis in states of caloric excess or cold exposure. Thus, in this article, we discuss the accumulating evidence suggestive of NNAT’s role in whole‐body metabolic regulation, while highlighting its potential to mediate adaptive thermogenesis via SERCA uncoupling.

Multiscale Modeling of Cardiovascular Function Predicts That the End-Systolic Pressure Volume Relationship Can Be Targeted via Multiple Therapeutic Strategies.

Most patients who develop heart failure are unable to elevate their cardiac output on demand due to impaired contractility and/or reduced ventricular filling. Despite decades of research, few effective therapies for heart failure have been developed. In part, this may reflect the difficulty of predicting how perturbations to molecular-level mechanisms that are induced by drugs will scale up to modulate system-level properties such as blood pressure. Computer modeling might help with this process and thereby accelerate the development of better therapies for heart failure. This manuscript presents a new multiscale model that uses a single contractile element to drive an idealized ventricle that pumps blood around a closed circulation. The contractile element was formed by linking an existing model of dynamically coupled myofilaments with a well-established model of myocyte electrophysiology. The resulting framework spans from molecular-level events (including opening of ion channels and transitions between different myosin states) to properties such as ejection fraction that can be measured in patients. Initial calculations showed that the model reproduces many aspects of normal cardiovascular physiology including, for example, pressure-volume loops. Subsequent sensitivity tests then quantified how each model parameter influenced a range of system level properties. The first key finding was that the End Systolic Pressure Volume Relationship, a classic index of cardiac contractility, was ∼50% more sensitive to parameter changes than any other system-level property. The second important result was that parameters that primarily affect ventricular filling, such as passive stiffness and Ca2+ reuptake via sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), also have a major impact on systolic properties including stroke work, myosin ATPase, and maximum ventricular pressure. These results reinforce the impact of diastolic function on ventricular performance and identify the End Systolic Pressure Volume Relationship as a particularly sensitive system-level property that can be targeted using multiple therapeutic strategies.

Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo-electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+-adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3 - states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.

The role of phospholamban and GSK3 in regulating rodent cardiac SERCA function.

Cardiac contractile function is largely mediated by the regulation of Ca2+ cycling throughout the lifespan. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is paramount to cardiac Ca2+ regulation, and it is well established that SERCA dysfunction pathologically contributes to cardiomyopathy and heart failure. Phospholamban (PLN) is a well-known inhibitor of the SERCA pump and its regulation of SERCA2a-the predominant cardiac SERCA isoform-contributes significantly to proper cardiac function. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase involved in several metabolic pathways, and we and others have shown that it regulates SERCA function. In this mini-review, we highlight the underlying mechanisms behind GSK3's regulation of SERCA function specifically discussing changes in SERCA2a and PLN expression and its potential protection against oxidative stress. Ultimately, these recent findings that we discuss could have clinical implications in the treatment and prevention of cardiomyopathies and heart failure.

Abstract 317: Endoplasmic Reticulum Stress Alters the Localization of the Prolyl Hydroxylase Ogfod1 in Adult Mouse Cardiomyocytes

Prolyl hydroxylation is a post-translational modification that is known to regulate several key cell functions including translation and protein stability. Enzymes that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase (2OGDO) family. A newly-identified member of the 2OGDO enzyme family, 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), catalyzes prolyl hydroxylation of ribosomal protein s23 (Rps23), a component of the 40S ribosome. To establish an in vivo role for Ogfod1, we ablated Ogfod1 in mice and subjected isolated perfused hearts to ischemia and reperfusion and found Ogfod1 ablation to be cardioprotective. The mechanisms by which these changes occur are currently unknown, so we investigated Ogfod1 regulation and mechanisms of cardioprotection. Previous work has shown that Ogfod1-mediated Rps23 prolyl hydroxylation occurs in the nucleus, where ribosomes are assembled. Additionally, Ogfod1 localizes to cytoplasmic stress granules in transformed cells exposed to endoplasmic reticulum (ER) stress. Based on these studies, we tested the hypothesis that Ogfod1 localization, and subsequently its function, change in response to stress. We induced ER stress by treating isolated adult cardiomyocytes with thapsigargin, and monitored Ogfod1 localization using immunofluorescence. Thapsigargin inhibits sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) which activates the unfolded protein response. In the absence of thapsigargin, Ogfod1 localized to the nucleus, peri-nuclear space, cytoplasm, and cell membrane. After stress induction, Ogfod1 nuclear- and perinuclear-localization decreased. This suggests that in murine adult cardiomyocytes subjected to ER stress, Ogfod1 is exported from the nucleus, potentially as a mechanism for down-regulating its activity. As Ogfod1 ablation has been found to be cardioprotective in mice, understanding Ogfod1 localization and regulation in the cardiomyocyte stress response may provide mechanistic insight that will be useful in developing treatments for stressors such as heart failure.

Transcriptomic Profiling of Ca2+ Transport Systems During the Formation of the Cerebral Cortex in Mice.

Cytosolic calcium (Ca2+) transients control key neural processes, including neurogenesis, migration, the polarization and growth of neurons, and the establishment and maintenance of synaptic connections. They are thus involved in the development and formation of the neural system. In this study, a publicly available whole transcriptome sequencing (RNA-Seq) dataset was used to examine the expression of genes coding for putative plasma membrane and organellar Ca2+-transporting proteins (channels, pumps, exchangers, and transporters) during the formation of the cerebral cortex in mice. Four ages were considered: embryonic days 11 (E11), 13 (E13), and 17 (E17), and post-natal day 1 (PN1). This transcriptomic profiling was also combined with live-cell Ca2+ imaging recordings to assess the presence of functional Ca2+ transport systems in E13 neurons. The most important Ca2+ routes of the cortical wall at the onset of corticogenesis (E11–E13) were TACAN, GluK5, nAChR β2, Cav3.1, Orai3, transient receptor potential cation channel subfamily M member 7 (TRPM7) non-mitochondrial Na+/Ca2+ exchanger 2 (NCX2), and the connexins CX43/CX45/CX37. Hence, transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1), transmembrane protein 165 (TMEM165), and Ca2+ “leak” channels are prominent intracellular Ca2+ pathways. The Ca2+ pumps sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and plasma membrane Ca2+ ATPase 1 (PMCA1) control the resting basal Ca2+ levels. At the end of neurogenesis (E17 and onward), a more numerous and diverse population of Ca2+ uptake systems was observed. In addition to the actors listed above, prominent Ca2+-conducting systems of the cortical wall emerged, including acid-sensing ion channel 1 (ASIC1), Orai2, P2X2, and GluN1. Altogether, this study provides a detailed view of the pattern of expression of the main actors participating in the import, export, and release of Ca2+. This work can serve as a framework for further functional and mechanistic studies on Ca2+ signaling during cerebral cortex formation.

SERCA overexpression reduces reperfusion-mediated cardiac microvascular damage through inhibition of the calcium/MCU/mPTP/necroptosis signaling pathways

Endothelial cells lining the microvasculature are particularly vulnerable to the deleterious effects of cardiac ischemia/reperfusion (I/R) injury, a susceptibility that is partially mediated by dysregulated intracellular calcium signals. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) functions to recycle calcium from the cytosol back to the endoplasmic reticulum. The purpose of this study is to explore the roles and mechanisms of SERCA in protecting microcirculation against cardiac I/R injury. Our data showed that overexpression of SERCA significantly reduced I/R-induced luminal stenosis and vascular wall edema, possibly through normalization of the ratio between eNOS and ET-1. I/R-induced erythrocyte morphological changes in micro-vessels could be reversed by SERCA overexpression through transcriptional inhibition of the expression of adhesive factors. In addition, SERCA-sustained endothelial barrier integrity reduced the likelihood of inflammatory cells infiltrating the myocardium. Furthermore, we found that SERCA overexpression attenuated intracellular calcium overload, suppressed mitochondrial calcium uniporter (MCU) expression, and prevented the abnormal opening of mitochondrial permeability transition pores (mPTP) in I/R-treated cardiac microvascular endothelial cells (CMECs). Interestingly, the administration of calcium activator or MCU agonist induced endothelial necroptosis in vitro and thus abolished the microvascular protection afforded by SERCA in reperfused heart tissue in vivo. In conclusion, by using gene delivery strategies to specifically target SERCA in vitro and in vivo, we identify a potential novel pathway by which SERCA overexpression protects microcirculation against cardiac I/R injury in a manner dependent on the calcium/MCU/necroptosis pathway. These findings should be taken into consideration in the development of pharmacological strategies for therapeutic interventions against cardiac microvascular I/R injury.

NAADP/SERCA3-Dependent Ca2+ Stores Pathway Specifically Controls Early Autocrine ADP Secretion Potentiating Platelet Activation.

Ca2+ signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca2+-ATPases) pump Ca2+ into internal stores that play a major role in the cytosolic Ca2+ concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca2+ mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions. To uncover the signaling mechanisms associated with Ca2+ mobilization from SERCA3-dependent stores leading to ADP secretion. Using platelets from wild-type or Serca3-deficient mice, we demonstrated that an early (within 5-10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca2+ is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca2+ mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca2+ mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3. Upon activation, an NAADP/SERCA3 Ca2+ mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca2+ stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca2+ mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.

Schistosoma mansoni sarco/endoplasmic reticulum Ca2+ ATPases (SERCA): role in reduced sensitivity to praziquantel.

Praziquantel leads to increase Ca2+ influx and disrupts Ca2+ homeostasis in adult Schistosoma. However, calcium influx is only one component in a series of molecular events leading to the drug effect and some downstream constituents of the cascade that is initiated by this interaction differ between worms with different degrees of susceptibility to praziquantel. Extensive use of the drug raises the concern regarding the selection of drug resistant parasites. SERCA participates in maintenance of Ca2+ homeostasis. Up-regulation of SERCA has been found in Schistosoma mansoni worms with reduced sensitivity to praziquantel. This could be due to increase cytosolic Ca2+, activation of calmodulin kinase II or may be due to SR/ER stress generated from oxidative stress that leads to impaired protein degradation. The significance of SERCA up-regulation is related to counter action of the drug effect by increasing the worm capacity to restore Ca2+ homeostasis, reducing cytosolic Ca2+ followed by lowering mitochondria Ca2+ and consequently inhibition of apoptosis beside its relation to P-glycoprotein. In schistosomes with reduced sensitivity to praziquantel, the agitations produced by Ca2+ influx and the downstream component of the cascade that is initiated by this interaction may be opposed by up-regulation of SERCA and possibly by certain elements of Ca2+ signaling which modulate the process determining cells entrance in the apoptotic state. Revealing the principal mechanisms of up-regulation of SERCA and its significance in reducing the effect of the drug could lead to possible strategies to reverse drug resistance or develop alternative therapies.

Dibutyl phthalate rapidly alters calcium homeostasis in the gills of Danio rerio

This study investigates the impacts of exposure to an environment Ca2+ challenge and the mechanism of action of dibutyl phthalate (DBP) on Ca2+ influx in the gills of Danio rerio. In vitro profile of 45Ca2+ influx in gills was verified through the basal time-course. Fish were exposed to low, normal and high Ca2+ concentrations (0.02, 0.7 and 2 mM) for 12 h. So, gills were morphologically analysed and ex vivo45Ca2+ influx at 30 and 60 min was determined. For the in vitro studies, gills were treated for 60 min with DBP (1 pM, 1 nM and 1 μM) with/without blockers/activators of ionic channels, Ca2+ chelator, inhibitors of ATPases, ionic exchangers and protein kinase C to study the mechanism of DBP-induced 45Ca2+ influx. Exposure to high environmental Ca2+ augmented 45Ca2+ influx when compared to fish exposed to normal and low Ca2+ concentrations. Additionally, histopathological changes were observed in the gills of fish maintained for 12 h in low and high Ca2+. In vitro exposure of gills to DBP (1 pM) disturbed Ca2+ homeostasis. DBP stimulated 45Ca2+ influx in gills through the transitory receptor potential vanilloid 1 (TRPV1), and reverse-mode Na+/Ca2+ exchanger (NCX) activation, protein kinase C and K+ channels and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). These data suggest that in vivo short-term exposure of gills to low and high Ca2+ leads to 45Ca2+ influx and histopathological changes. Additionally, the DBP-induced rapid 45Ca2+ influx is mediated by TRPV1, NCX activation with the involvement of PKC, K+-channels and SERCA, thereby altering Ca2+ homeostasis.

The phospholamban p.(Arg14del) pathogenic variant leads to cardiomyopathy with heart failure and is unresponsive to standard heart failure therapy

Phospholamban (PLN) plays a role in cardiomyocyte calcium handling as primary inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The p.(Arg14del) pathogenic variant in the PLN gene results in a high risk of developing dilated or arrhythmogenic cardiomyopathy with heart failure. There is no established treatment other than standard heart failure therapy or heart transplantation. In this study, we generated a novel mouse model with the PLN-R14del pathogenic variant, performed detailed phenotyping, and tested the efficacy of established heart failure therapies eplerenone or metoprolol. Heterozygous PLN-R14del mice demonstrated increased susceptibility to ex vivo induced arrhythmias, and cardiomyopathy at 18 months of age, which was not accelerated by isoproterenol infusion. Homozygous PLN-R14del mice exhibited an accelerated phenotype including cardiac dilatation, contractile dysfunction, decreased ECG potentials, high susceptibility to ex vivo induced arrhythmias, myocardial fibrosis, PLN protein aggregation, and early mortality. Neither eplerenone nor metoprolol administration improved cardiac function or survival. In conclusion, our novel PLN-R14del mouse model exhibits most features of human disease. Administration of standard heart failure therapy did not rescue the phenotype, underscoring the need for better understanding of the pathophysiology of PLN-R14del-associated cardiomyopathy. This model provides a great opportunity to study the pathophysiology, and to screen for potential therapeutic treatments.

Thapsigargins and induced chemical defence in Thapsia garganica

Thapsigargin and related compounds are produced by Thapsia garganica L. (Apiaceae) and are thought to be a defence compound against herbivory. Thapsigargin inhibits the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) in both vertebrates and invertebrates. This activity is responsible for its potent toxicity, as well as the potential use to treat solid tumours. However, the ecological role and regulation of thapsigargin are not well understood, and the aim of this study was to investigate if thapsigargin biosynthesis was responsive to leaf damage. To test the response to potential leaf damage during a herbivory, greenhouse plants were subjected to clipping to mimic the physical damage. Unclipped versus clipped plants were sampled for chemical analysis and the gene expression for the two known thapsigargin biosynthetic genes (TgTPS2 and TgCYP76AE2) was investigated. Data obtained by LC–ESI–MS/MS were used to perform molecular networking to identify chemical constituents related to thapsigargin and its biosynthesis. The results show a significant change in a plant’s chemical profile after mimicking an herbivory event. Both the chemical analysis and gene expression data show that T. garganica plants can induce the biosynthesis of this class of defence compounds at the site of an attack. Thapsigargins are clearly the dominant defence compounds in these plants, and they seem to be produced through a common biosynthetic pathway with little diversity. This likely means that T. garganica has a relatively simple response to herbivory, as opposed to many other plant species that have been shown to have complex metabolite responses to herbivory.

Is there a role for sarcolipin in avian facultative thermogenesis in extreme cold?

Endotherms defend their body temperature in the cold by employing shivering (ST) and/or non-shivering thermogenesis (NST). Although NST is well documented in mammals, its importance to avian heat generation is unclear. Recent work points to a prominent role for the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) in muscular NST. SERCA's involvement in both ST and NST, however, posits a tradeoff between these two heat-generating mechanisms. To explore this tradeoff, we assayed pectoralis gene expression of adult songbirds exposed to chronic temperature acclimations. Counter to mammal models, we found that cold-acclimated birds downregulated the expression of sarcolipin (SLN), a gene coding for a peptide that promotes heat generation by uncoupling SERCA Ca2+ transport from ATP hydrolysis, indicating a reduced potential for muscular NST. We also found differential expression of many genes involved in Ca2+ cycling and muscle contraction and propose that decreased SLN could promote increased pectoralis contractility for ST. Moreover, SLN transcript abundance negatively correlated with peak oxygen consumption under cold exposure (a proxy for ST) across individuals, and higher SLN transcript abundance escalated an individual's risk of hypothermia in acute cold. Our results therefore suggest that SLN-mediated NST may not be an important mechanism of-and could be a hindrance to-avian thermoregulation in extreme cold.

48-OR: Pancreatic ß Cell SERCA2 Deficiency Leads to Impaired Proinsulin Processing and Reduced ER to Golgi Protein Trafficking

Impaired proinsulin processing is observed in both type 1 and type 2 diabetes. We have previously shown that reductions in endoplasmic reticulum (ER) calcium (Ca2+) in the pancreatic β cell arising from impaired activity of the Sarcoendoplasmic Reticulum Ca2+ ATPase (SERCA) pump are associated with increased proinsulin secretion. However, the mechanisms responsible for reduced proinsulin processing in the context of SERCA2 deficiency remain incompletely understood. To test this, we developed mice with β cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance and reduced glucose-stimulated insulin secretion without evidence of impaired insulin sensitivity. ER Ca2+ levels in islets from βS2KO mice were significantly reduced, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2 and CPE. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Consistent with this, trafficking assays were performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Moreover, pancreas sections from βS2KO mice showed increased co-localization of proinsulin and ProPC2 in the early compartments of the secretory pathway. Taken together, these data suggest that loss of SERCA2 activity and ER Ca2+ loss in the pancreatic β cell leads to impaired proinsulin processing via reduced maturation and trafficking of proinsulin processing enzymes. Disclosure T. Kono: None. H. Iida: None. T.S. Jarvela: None. C. Lee: None. X. tong: None. P. Arvan: None. I. Lindberg: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institutes of Health (R01DK093954); U.S. Department of Veterans Affairs (2-I01BX001733)

IRE1\u03b1 and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into “responder” and “non-responder”. By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

Acute exposure to bis(2-ethylhexyl)phthalate disrupts calcium homeostasis, energy metabolism and induces oxidative stress in the testis of Danio rerio

Bis(2-ethylhexyl)phthalate (BEHP) negatively affects testicular functions in different animal species, disturbing reproductive physiology and male fertility. The present study investigated the in vitro acute effect of BEHP on the mechanism of action of ionic calcium (Ca2+) homeostasis and energy metabolism. In addition, the effect of BEHP on oxidative stress was studied in vitro and in vivo in the testis of Danio rerio (D. rerio). Testes were treated in vitro for 30 min with 1 μM BEHP for 45Ca2+ influx measurements. Testes were also incubated with 1 μM BEHP for 1 h (in vitro) or 12 h (in vivo) for the measurements of lactate content, 14C-deoxy-d-glucose uptake, lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT) activity, total reactive oxygen species (ROS) production and lipid peroxidation. In addition, the effect of BEHP (1 μM) on GGT, glutamic oxaloacetic transferase (GOT) and glutamic pyruvic transferase (GPT) activity in the liver was evaluated after in vivo treatment for 12 h. BEHP disturbs the Ca2+ balance in the testis when given acutely in vitro. BEHP stimulated Ca2+ influx occurs through L-type voltage-dependent Ca2+ channels (L-VDCC), transitory receptor potential vaniloid (TRPV1) channels, reverse-mode Na+/Ca2+ exchanger (NCX) activation and inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). BEHP affected energy metabolism in the testis by decreasing the lactate content and LDH activity. In vitro and in vivo acute effects of BEHP promoted oxidative stress by increasing ROS production, lipid peroxidation and GGT activity in the testis. Additionally, BEHP caused liver damage by increasing GPT activity.