Atomic-Level Mechanisms for Phospholamban Regulation of the Calcium Pump

AbstractWe performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca2+-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca2+-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1–7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K+ and 3 mM Mg2+. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K+ ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K+ in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H+771. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca2+-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump

A novel FRET-based screen in high-throughput format to identify inhibitors of malarial and human glucose transporters

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z′ factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC(50)) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening

Coding sequences of sarcoplasmic reticulum calcium ATPase regulatory peptides and expression of calcium regulatory genes in recurrent exertional rhabdomyolysis

Background: Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). Objectives: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. Animals: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. Methods: Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real-time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). Results: The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex-by-phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P \u3c .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. Conclusions and Clinical Importance: The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1

The Capital Structure and Governance of a Mortgage Securitization Utility

We explore the capital structure and governance of a mortgage-insuring securitization utility operating with government reinsurance for systemic or 'tail' risk. The structure we propose for the replacement of the GSEs focuses on aligning incentives for appropriate pricing and transfer of mortgage risks across the private sector and between the private sector and the government. We present the justification and mechanics of a vintage-based capital structure, and assess the components of the mortgage guarantee fee, whose size we find is most sensitive to the required capital ratio and the expected return on that capital. We discuss the implications of selling off some of the utility's mortgage credit risk to the capital markets and how the informational value of such transactions may vary with the level of risk transfer. Finally, we explore how mutualization could address incentive misalignments arising out of securitization and government insurance, as well as how the governance structure for such a financial market utility could be designed

Co-reconstitution of Phospholamban Mutants with the Ca-ATPase Reveals Dependence of Inhibitory Function on Phospholamban Structure*

in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by �-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 °C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetrame