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In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca 2؉ -transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca 2؉ in activation of the overall reaction due to a decreased E 2 to E 1 Ca 2 transition rate and an increased rate of Ca 2؉ dissociation from E 1 Ca 2 . At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca 2؉ level (either an enhanced efflux of lumenal Ca 2؉ through the pump in E 2 form or insensitivity to inhibition by lumenal Ca 2؉ ). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca 2؉ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E 2 P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.Sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) 1 are single-subunit membrane-spanning P-type ATPases that mediate the uphill transport of cytoplasmic Ca 2ϩ into the lumen of intracellular stores with a stoichiometry of two Ca 2ϩ per ATP hydrolyzed (1-3). Studies of the SERCA1a enzyme have shown that Ca 2ϩ transport and ATP utilization are coupled through long-range intramolecular interactions between the 10-helix transmembrane domain containing the Ca 2ϩ binding sites and the cytoplasmic actuator, phosphorylation, and nucleotidebinding domains (1-4). In the reversible catalytic cycle (5), the binding of the two calcium ions with high affinity from the cytoplasmic side of the membrane triggers the phosphorylation of Asp 351 by ATP, whereas the dephosphorylation occurs when the translocated calcium ions have been released from lowaffinity lumenally facing sites in exchange with protons being countertransported (Scheme 1).The SERCAs have long been known as key enzymes in the cytoplasmic Ca 2ϩ signaling events, and more recent studies have also drawn attention to the importance of the luminal Ca 2ϩ content for Ca 2ϩ signaling, folding and processing of newly synthesized proteins, and control of cell growth and apoptosis (6 -9). In addition to the functionally well characterized SERCA1a isoform, the 3 human SERCA genes encode at least 8 other SERCA proteins by alternative splicing (10 -15). Whereas SERCA2b seems to be a ubiquitously expressed housekeeping enzyme, the other isoforms are limited to specific c...

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Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.