The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

Blockade of Oncogenic NOTCH1 with the SERCA Inhibitor CAD204520 in T Cell Acute Lymphoblastic Leukemia.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).

Nonlinear relationship between ER Ca2+ depletion versus induction of the unfolded protein response, autophagy inhibition, and cell death.

Endoplasmic reticulum (ER) Ca2+ depletion activates the unfolded protein response (UPR), inhibits bulk autophagy and eventually induces cell death in mammalian cells. However, the extent and duration of ER Ca2+ depletion required is unknown. We instigated a detailed study in two different cell lines, using sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors to gradually reduce ER Ca2+ levels in a controlled manner. Remarkably, UPR induction (as assessed by expression analyses of UPR-regulated proteins) and autophagy inhibition (as assessed by analyses of effects on starvation-induced bulk autophagy) required substantially higher drug concentrations than those needed to strongly decrease total ER Ca2+ levels. In fact, even when ER Ca2+ levels were so low that we could hardly detect any release of Ca2+ upon challenge with ER Ca2+ purging agents, UPR was not induced, and starvation-induced bulk autophagy was still fully supported. Moreover, although we observed reduced cell proliferation at this very low level of ER Ca2+, cells could tolerate prolonged periods (days) without succumbing to cell death. Addition of increasing concentrations of extracellular EGTA also gradually depleted the ER of Ca2+, and, as with the SERCA inhibitors, EGTA-induced activation of UPR and cell death required higher EGTA concentrations than those needed to strongly reduce ER Ca2+ levels. We conclude that ER Ca2+ depletion-induced effects on UPR, autophagy and cell death require either an extreme general depletion of ER Ca2+ levels, or Ca2+ depletion in areas of the ER that have a higher resistance to Ca2+ drainage than the bulk of the ER.

Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response.

Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 µm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

Coupling Ratio for Ca(2+) Transport by Calcium Oxalate Precipitation.

The SERCA isoform 1a is constructed to transport 2 Ca(2+) ions across the sarcoplasmic reticulum membrane coupled to the hydrolysis of one molecule of MgATP. However, observed coupling ratios for Ca(2+) transported/ATP hydrolzyed are usually less than 2:1, since part of the Ca(2+) accumulated at high intravesicular concentrations by the active transport of Ca(2+) leaks out of the vesicles because of Ca(2+)-induced Ca(2+) exchange. However, in the presence of a high concentration of oxalate (5 mM) Ca(2+) will precipitate as Ca-oxalate inside the vesicles and thereby be prevented from leaking out and, in addition, this treatment will reduce the intravesicular free concentration of Ca(2+) to a level where optimal coupling ratios of 2:1 can be achieved.

Ca(2+) Binding and Transport Studied with Ca(2+)/EGTA Buffers and (45)Ca(2+).

The chapter describes procedures useful for determination of Ca(2+) binding by membranous Ca(2+)-ATPase based on the correction for the removal of Ca(2+) present in a non-bound state in the suspension medium. This is done by a filtration procedure that retains the membranous material on the Millipore filters. With suitable sucking devices it is possible to gently remove without dehydration nearly all medium from the Ca(2+) containing membranes, except that required for wetting of the filters on which they are deposited. Correction for this effect can be done with a double-filter where the radioactive content of the lower (protein-free) filter is subtracted from that present in the upper filter for calculation of Ca(2+) binding. This methodology can be used to study the effect of inhibitors on Ca(2+) binding and -transport, and with Ca(2+)/EGTA buffers to explore the Ca(2+) binding affinities and cooperative aspects of the two transport sites.

Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 µM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium.

The inhibition of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) by thapsigargin (Tg) and Tg-type analogues is considered to trigger cell death by activation of apoptotic pathways. Some of these analogues may be useful as antineoplastic agents after appropriate targeting as peptide conjugated prodrugs to cancer cells. With this in mind, this study evaluates the effect on LNCaP androgen-sensitive cancer cells of thapsigargin substituted with 12-aminododecanoyl linkers and Leu (Leu-8ADT), aspartate (Asp-8ADT) or Boc-8ADT. Our results show that both Leu-8ADT and Asp-8ADT result in rapid ER calcium depletion and an influx of calcium across the plasma membrane by activation of store-operated calcium entry. By contrast, ER Ca(2+) depletion by Boc-8ADT is a very slow process that does not perceptibly increase cytosolic Ca(2+) and activate store-operated calcium entry, because the inhibition of SERCA with this compound is very slow. Nevertheless, we find that Boc-8ADT is a more efficient inducer of apoptosis than both Tg and Leu-8ADT. Compared with Tg and the other analogues, apoptosis induced by Asp-8ADT is very modest, although this compound also activates store-operated calcium entry and at high concentrations (1 µm) causes severe morphological changes, reflecting decreased cell viability. We conclude that many factors need to be considered for optimization of these compounds in antineoplastic drug design. Among these ER stress induced by Ca(2+) endoplasmic reticulum mobilization seems particularly important, whereas the early cytosolic increase of Ca(2+) concentration preceding the executive phase of apoptosis appears to be of no, or little, consequence for a subsequent apoptotic effect.

Water-mediated interactions influence the binding of thapsigargin to sarco/endoplasmic reticulum calcium adenosinetriphosphatase.

A crystal structure suggests four water molecules are present in the binding cavity of thapsigargin in sarco/endoplasmic reticulum calcium ATPase (SERCA). Computational chemistry indicates that three of these water molecules mediate an extensive hydrogen-bonding network between thapsigargin and the backbone of SERCA. The orientation of the thapsigargin molecule in SERCA is crucially dependent on these interactions. The hypothesis has been verified by measuring the affinity of newly synthesized model compounds, which are prevented from participating in such water-mediated interactions as hydrogen-bond donors.

Active detergent-solubilized H+,K+-ATPase is a monomer.

The H(+),K(+)-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a ß-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H(+),K(+)-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C(12)E(8), followed by exchange of C(12)E(8) with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the ß-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s(20,)(w) = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H(+),K(+)-ATPase α,ß-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,ß-protomer with 0.9-1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α(2),ß(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, α,ß H(+),K(+)-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca(2+)-ATPase and Na(+),K(+)-ATPase.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.

Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

The Plasmodium falciparum Ca(2+)-ATPase PfATP6: insensitive to artemisinin, but a potential drug target.

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139 kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.

Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases.

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Purified E255L mutant SERCA1a and purified PfATP6 are sensitive to SERCA-type inhibitors but insensitive to artemisinins.

The antimalarial drugs artemisinins have been described as inhibiting Ca(2+)-ATPase activity of PfATP6 (Plasmodium falciparum ATP6) after expression in Xenopus oocytes. Mutation of an amino acid residue in mammalian SERCA1 (Glu(255)) to the equivalent one predicted in PfATP6 (Leu) was reported to induce sensitivity to artemisinin in the oocyte system. However, in the present experiments, we found that artemisinin did not inhibit mammalian SERCA1a E255L either when expressed in COS cells or after purification of the mutant expressed in Saccharomyces cerevisiae. Moreover, we found that PfATP6 after expression and purification from S. cerevisiae was insensitive to artemisinin and significantly less sensitive to thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone than rabbit SERCA1 but retained higher sensitivity to cyclopiazonic acid, another type of SERCA1 inhibitor. Although mammalian SERCA and purified PfATP6 appear to have different pharmacological profiles, their insensitivity to artemisinins suggests that the mechanism of action of this class of drugs on the calcium metabolism in the intact cell is complex and cannot be ascribed to direct inhibition of PfATP6. Furthermore, the successful purification of PfATP6 affords the opportunity to develop new antimalarials by screening for inhibitors against PfATP6.

Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs.

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

P-type ATPases as drug targets: tools for medicine and science.

P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.

Protein-protein contacts in solubilized membrane proteins, as detected by cross-linking.

The amount of detergent required for the solubilization of membrane proteins needs to be optimised as an excess may cause loss of activity and insufficiency may result in poor solubilization or heterogeneous samples. With sarcoplasmic reticulum Ca2+ -ATPase as an example we show by cross-linking that it can be misleading to choose the proper amount of detergent based on clarification of membrane suspensions, because clarification -as detected by turbidity measurements, for instance- precedes full protein solubilization as monomers. We demonstrate that to assess the extent of sample homogeneity at a given detergent/protein ratio, cross-linking followed by HPLC gel filtration in detergent usefully complements cross-linking followed by SDS-PAGE.

Natural products as starting materials for development of second-generation SERCA inhibitors targeted towards prostate cancer cells.

An analysis of the binding of the 8-O-N-tert-butoxycarbonyl-12-aminododecanoyl derivative of 8-O-debutanoylthapsigargin to the target molecule, the SERCA pump, has revealed the importance of the length and flexibility of the side chain attached to O-8. Based on the analysis a series of analogues to the 2-unsubstituted analogue trilobolide has been constructed and shown to be equipotent with thapsigargin as SERCA inhibitors. Only the 12-Boc-aminododecaonoyl derivative, however, was found to be apoptotic.