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|Title:||Caloxins: A Novel Class of Plasma Membrane Ca2+ Pump Inhibitors|
|Keywords:||Ionized Calcium, Ca2+, Plasma, membrane, pump, inhibitors,|
|Abstract:||Ionized calcium (Ca2+) is a signaling messenger that controls numerous cellular processes essential for life. The fidelity of Ca2+ signaling depends on the mechanisms that dynamically regulate its cytosolic concentration and maintain it at a low level in a resting cell. Plasma Membrane Ca2+ ATPase (PMCA) is a high affinity Ca2+ extrusion pathway involved in Ca2+ homeostasis and signal transduction. PMCA are encoded by 4 genes (PMCA1-4), which are expressed in a tissue dependent manner. The diversity of PMCA isoforms is further increased by alternative splicing. Changes in PMCA activity occur in heart failure and hypertension. Specific inhibitors of other ion transporters such as thapsigargin and digoxin, have made their mark in cell biology, but the currently used inhibitors of PMCA (vanadate and eosin) are non-specific. Thus, selective inhibitors of PMCA are needed to discern its role in Ca2+ signaling in physiology and pathophysiology. We introduced the concept of caloxins - peptides that specifically inhibit the activity of PMCA by binding to one of its five extracellular domains (exdoms) 1 to 5. The earlier caloxins including 2a1 and 3a1 were obtained by screening a phage display random 12-amino acid peptide (Ph.D-12) library for binding to synthetic peptides based on the exdom sequences. However, they all had low affinity. The objective of this research was to develop caloxins with high affinity and PMCA4 isoform selectivity. A two-step screening method was developed to screen the Ph.D-12 library to first bind to the synthetic exdom of PMCA4, followed by affinity chromatography using PMCA protein purified from human erythrocyte ghosts (mainly PMCA4). This method was used to obtain caloxins 1b1 and 1b2 to bind to the N and C-terminal halves of the exdom 1 of PMCA4, respectively. Both caloxins 1b1 and 1b2 had a 10-fold higher affinity than the prototype caloxin 2a1 and showed slight PMCA4 isoform preference. To engineer inhibitors with greater affinity and PMCA4 isoform selectivity, Ph.D caloxin 1b1 like peptide library was constructed. Most of the peptides expressed in this library differed from caloxin 1b1 in 0, 1, 2 or 3 amino acid residues at random. The library was screened to obtain several peptides one of which was caloxin 1c2. Caloxin 1c2 had 200-fold higher affinity than caloxin 2a1 and was isoform selective, with greater than 10-fold affinity for PMCA4 than for PMCA isoforms 1, 2 or 3. Thus, caloxin 1c2 is the first high affinity PMCA inhibitor that also is selective for an individual PMCA isoform. The second aim of this research was to establish that caloxin 1c2 binds to PMCA protein in erythrocyte ghosts. Two photoreactive caloxin 1c2-derivatives containing the photoactivable residue benzoylphenylalanine (Bpa) and a C-terminal biotin tag were used. Bpa substituted tryptophan at position 3 (3Bpa1c2-biotin) and serine at position 16 (16Bpa1c2-biotin) in caloxin 1c2. Both the derivatives inhibited PMCA activity in the erythrocyte ghosts. The intensity of the biotin label in the photolabeled erythrocyte ghosts was much stronger with 3Bpa1c2-biotin, which was then used in the subsequent experiments. The photolabeled proteins in erythrocyte ghosts were detected as a 250-270 kDa doublet in Western blots using streptavidin and the PMCA specific antibody. The degree of photolabeling depended on the UV-crosslinking time, and on the concentrations of 3Bpa1c2-biotin and the ghost protein. The selectivity of the photolabeling site was confirmed by decreased photolabel incorporation at 250-270 kDa doublet in the presence of excess caloxin 1c2 and the synthetic exdom 1X peptide of PMCA4. The photolabeled erythrocyte ghosts were solubilized and analyzed by immunoprecipitation with the PMCA specific antibody. The immunoprecipitate showed a 250-270 kDa doublet in Western blots using streptavidin. This confirmed that PMCA protein was photolabeled by the photoreactive derivatives of caloxin 1c2. Thus, caloxin 1c2 inhibits PMCA activity by binding to the exdom 1X of PMCA4. My work in M.Sc. initiated the concept of caloxins in the literature. This research has taken it to the stage where we can obtain caloxins selective for individual PMCA isoforms. This contrasts with the relative paucity of inhibitors specific for individual isoforms of other ion pumps. The high affinity isoform selective caloxin 1c2 and previous caloxins are being used to study PMCA physiology in our lab and by other researchers. Since caloxins act when added extracellularly and it is possible to obtain PMCA isoform selective caloxins, it is anticipated that they will aid in understanding the role of PMCA in signal transduction and homeostasis in health and disease.|
|Appears in Collections:||Open Access Dissertations and Theses|
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