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Project

Purification of the Secretory Pathway Ca2+/Mn2+ ATPases for Functional Analysis and Drug Discovery

The organellar P2-type Ca2+-transport ATPases, namely Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Secretory Pathway Ca²⁺ ATPase (SPCA), are essential to maintain a low resting cytosolic Ca2+ level. At the same time they load respectively the intracellular endoplasmic reticulum (ER) and Golgi stores with Ca2+. This allows Ca2+ signaling to occur and it supports several organellar processes such as protein folding and maturation. Besides Ca2+, SPCA also transports Mn2+. The structure of SERCA1a has been solved by crystallography in different conformational states of the transport cycle. It serves as an excellent template for exploring and understanding the structural features of SPCA that shares 40% sequence similarity with SERCA. The study of SPCA pumps in cells is hampered because its enzymatic readouts are overshadowed by the stronger signals of the more active SERCA and PMCA Ca2+-ATPases. We therefore set up a purified, native-like proteoliposome system for the two SPCA isoforms SPCA1 and SPCA2, which was used throughout the two projects in this thesis.

In the first chapter, we showed that the SPCA1 ATPase activity was activated by various lipids, especially sphingomyelin and phosphatidic acid. Next, we explored the possibility to develop an SPCA1 specific inhibitor based on the SERCA1 specific inhibitor thapsigargin (Tg). To this end, we proved with the mutagenesis study that Tg binds to SPCA1 at a conserved pocket similar to SERCA1. Next, by comparing the variable inhibition potencies of Tg analogues on SPCA1, we characterized the structure-activity relationship (SAR) on SPCA1. Our results showed that the Tg sidechains O3, O8 and O10, that critically contribute to SERCA1 inhibition, are only weakly involved in SPCA1 inhibition. It is therefore possible to alter the specificity of Tg towards SPCA by modifying these SERCA-critical sidechains into more SPCA1 compatible groups.

In the second chapter, we compared side by side the Ca2+- and Mn2+- dependent SPCA1 and SPCA2 activities. Based on the ATPase and auto-phosphorylation assays, we determined the Ca2+ and Mn2+ affinities and turnover rates for both SPCA1 and SPCA2. We found that SPCA1 has a higher affinity for Mn2+ than for Ca2+, which is opposite from SPCA2. SPCA1 displays a higher Ca2+ turnover rate compared to Mn2+, while SPCA2 shows similar turnover rates for both substrates.

Unlike SPCA2, the Ca2+ and Mn2+-dependent autophosphorylation of SPCA1 presented a biphasic bihavior. Micromolar concentration of Ca2+ dramatically stimulated SPCA1 phospho-enzyme formation at low ATP concentration, while the stimulation by micromolar Mn2+ was moderate. Since the N-terminus of the yeast orthologue PMR1 contains a Ca2+ binding regulatory EF-hand-like motif, we investigated the role of this conserved region in SPCA1 by mutagenesis. The SPCA1 N-terminal mutants displayed reduced Ca2+ binding and a stronger increase in the affinity for Ca2+ than for Mn2+ in both the ATPase assay and the first phase of the phosphorylation assay. The latter suggests that a larger fraction of SPCA1 mutants compared to WT were active at a physiological Ca2+ concentration. At the same time, the relative magnitude of activation by the second phase was lowered. Taken together, these results suggest that the N-terminal region indeed plays a role in SPCA1 activation, in particular by Ca2+, and may at least partially be responsible for the different enzymatic activities between SPCA1 and SPCA2.

We also showed that the Ca2+ affinity of SPCA1 is sensitive to ATP concentrations in the reaction medium, and that higher amounts of ATP promote the phospho-enzyme formation at lower Ca2+ concentrations. Therefore, in a cellular context in the presence of millimolar ATP, SPCA1 activation can be achieved at physiologically relevant Ca2+ concentrations in a microdomain close to a Ca2+ channel. On the other hand, the combinatorial effect of ATP and Ca2+ on SPCA activity may come into action in keratinocytes, in which the cytosolic ATP level is lower compared to other cell types, whereas the cytosolic Ca2+ concentration is higher, e.g. after mechanical stimulation.

In conclusion, we showed that a purified and reconstituted system is a useful tool for the functional and pharmacological exploration of SPCA. More specifically, this thesis contributes to a better understanding of Tg inhibition of SPCA1 as well as the biochemical properties and differences in regulation of SPCA1/2.

Date:1 Oct 2012 →  14 Jun 2019
Keywords:Golgi, thapsigargin, calcium ATPase, regulation, membrane protein purification, calcium, reconstitution, manganese
Disciplines:Physiology
Project type:PhD project