Irene C. Mangialavori

A new conformation in sarcoplasmic reticulum calcium pump and plasma membrane Ca2+ pumps revealed by a photoactivatable phospholipidic probe.

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[125I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([125I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [125I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca2+. This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca2+ and calmodulin decreases by approximately the same amount. However, incubation with Ca2+ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [125I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E1 conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca2+ + C28 for SERCA and Ca2+ alone for PMCA), and one in which the enzyme is fully active (Ca2+ for SERCA and Ca2+-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.

Peptide Anchor for Folate-Targeted Liposomal Delivery

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction

Background: Plasma membrane calcium ATPases interact dynamically with the submembrane actin cytoskeleton. Results: Biophysical and functional assays show that purified plasma membrane calcium ATPase binds to G-actin and is activated by short actin oligomers. Conclusion: Plasma membrane calcium ATPases are regulated by polymerizing actin independently of regulation by calmodulin. Significance: Dynamic actin participates in cytosolic Ca2+ homeostasis by regulating plasma membrane calcium ATPase activity. As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis.

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca2+-pump (PMCA) in the membrane region upon interaction with Ca2+, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, measuring the shift of conformation E2 to the auto-inhibited conformation E1I and to the activated E1A state, titrating the effect of Ca2+ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca2+ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca2+ transport but does not modify the affinity for Ca2+ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E1I to the activated E1A conformation and thus modulating the transport of Ca2+. This is reflected in the different apparent constants for Ca2+ in the absence of CaM (calculated by Ca2+-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca2+ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca2+ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca2+ binding and activation.

Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.

Plasma Membrane Calcium Pump (PMCA) Differential Exposure of Hydrophobic Domains after Calmodulin and Phosphatidic Acid Activation

The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [125I]TID-PC/16 to the protein. In the presence of Ca2+ both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [125I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [125I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [125I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.

Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids: Effect of the Micelle-Vesicle Transition and the Bilayer Thickness

Background: Membrane proteins require phospholipids to be biologically active. Results: An increase of phosphatidylcholine/detergent molar ratio leads to a biphasic behavior of the PMCA Ca2+-ATPase activity, whose maximum depends on phosphatidylcholine characteristics. Conclusion: The optimum hydrophobic thickness for PMCA structure and Ca2+-ATPase activity is about 24 Å. Significance: Differential modulation by neutral phospholipids could be a general mechanism for regulating membrane protein function. The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca2+ pump (PMCA). We found that Ca2+-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca2+-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA.

Conformational changes produced by ATP binding to the plasma membrane calcium pump

Background: The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes. Results: We identified different conformations after the association of Ca2+, ATP, and vanadate to PMCA. Conclusion: PMCA forms a stable complex with Ca2+ and vanadate; ATP can bind to all pump conformations. Significance: This study found a new intermediate in the PMCA reaction cycle; all of the intermediates interact with ATP. The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Dynamic lipid-protein stoichiometry on E1 and E2 conformations of the Na+/K+ -ATPase.

Annular lipid-protein stoichiometry in native pig kidney Na+/K+ -ATPase preparation was studied by [125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in E2, i.e., the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E2 conformation.

A synthetic bioisoster of trimethadione and phenytoin elicits anticonvulsant effect, protects the brain oxidative damage produced by seizures and exerts antidepressant action in mice

Epilepsy is recognized as one of the most common and serious neurological disorder affecting 1-2% of the world׳s population. The present study demonstrates that systemic administration of 3-butyl-5,5-dimethyl-1,2,3-oxathiazolidine-4-one-2,2-dioxide (DIOXIDE), a synthetic compound bioisoster of trimethadione and phenytoin (classical anticonvulsants), elicits a dose dependent anticonvulsant response in mice submitted to the subcutaneous pentylenetetrazole seizure test (scPTZ). Among various factors supposed to play role in epilepsy, oxidative stress and reactive species have strongly emerged. The protection exerted by DIOXIDE over the extent of brain oxidative damage produced by PTZ was determined, by measuring the levels of lipid peroxidation and reduced glutathione and the activity of Na(+)/K(+)-ATPase. Psychiatric disorders represent frequent comorbidities in persons with epilepsy. In this report, the potential anxiolytic and antidepressant activities of DIOXIDE were evaluated in several widely used models for assessing anxiolytic and antidepressant activities in rodents. Although DIOXIDE did not evidence anxiolytic activity at the doses tested, it revealed a significant antidepressant-like effect. Preliminary studies of its mechanism of action, by means of its capacity to act via the GABAA receptor (using the [(3)H]flunitrazepam binding assay in vitro and the picrotoxin test in vivo) and the Na(+) channel (using the alkaloid veratrine, a voltage-Na(+) channel agonist) demonstrated that the anticonvulsant effect is not likely related to the GABAergic pathway and the antidepressant-like effect could be due to its Na(+) channel blocking properties. The results for DIOXIDE suggested it as a new anticonvulsant-antioxidant and antidepressant compound that deserves further development.