McCue, Hannah (2011) Characterisation of two calmodulin-related calcium sensors, CaBP7 and CaBP8. Doctoral thesis, University of Liverpool.
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The CaBPs are a family of small EF-hand-containing calcium binding proteins with limited homology to calmodulin (CaM). The family comprises seven genes with alternative splicing giving rise to a greater number of protein variants. Two distinct groups exist within the CaBP family based on differences in their cation binding properties and domain organization: CaBPs 1-5 and CaBP7 & 8. CaBPs 1-5 interact with a number of known CaM target proteins but generally exert unique regulatory roles and have been implicated in the regulation of numerous Ca2+-channels. A number of interacting binding partners have also been identified which do not overlap with those of CaM and knockout studies have revealed non-redundant roles for CaBP4 and CaBP5. Much less is known about the functions of CaBP7 and CaBP8 with only a direct interaction with phosphatidylinositol 4-kinase III β (PI4KIIIβ) having been characterized in detail. The research presented in this thesis focused on the less well characterised members of this protein family, CaBP7 and CaBP8. Bioinformatic analyses revealed that CaBP7 and CaBP8 form a distinct group of calcium sensors from the rest of the CaBP family and are highly conserved throughout evolution. Examination of CaBP sequences present in species representing important evolutionary nodes indicated that the CaBP family first arose with the appearance of early vertebrates. The primordial CaBP family members, CaBP1S and CaBP8, were identified in lamprey, the closest living relative to the common vertebrate ancestor. The expansion in the number of CaBP proteins from lamprey to mammals largely coincides with a whole genome duplication event occurring before the divergence of cartilaginous fish. A transient expansion of the group encompassing CaBPs1-5 between the divergence of cartilaginous fish and the emergence of bony fish is also apparent. Examination of the targeting mechanisms of CaBP7 and CaBP8 demonstrated that these proteins form a unique class of calcium sensors which associate with cellular membranes via a C-terminal tail anchor. The membrane topology of CaBP7 and CaBP8 was found to be characteristic of the tail anchored class of integral membrane proteins and was consistent with the previously reported interaction with the cytosolic enzyme PI4KIIIβ. Further examination of their subcellular distributions revealed that CaBP7 and CaBP8 localise to the trans-Golgi network and to vesicles which were positive for markers of the late endosomal and lysosomal pathway. This is the first reported instance of a CaM-related calcium sensor localising to membranes of acidic compartments. CaBP7 also co-localised with markers of late endosomes and lysosomes in dividing cells. These CaBP7 positive vesicles redistributed to the vicinity of the interzonal microtubules and opposite poles of the spindle during cytokinesis. In accordance with previous reports, knockdown of CaBP7 in HeLa cells resulted in an increase in the number of binucleate cells, inferring that CaBP7 plays an important role in the regulation of cytokinesis. CaBP7 was shown to co-localise with VAMP7, a protein known to be important for Ca2+-regulated lysosomal exocytosis and also for the completion of cytokinesis. In addition CaBP7 localised to VSVG positive vesicles which have been previously shown to directly dock and fuse with the plasma membrane at the midzone. Lysosomes are emerging as important platforms for Ca2+-signalling, and proteins important for the release of lysosomal Ca2+ stores have also been implicated in mitosis. This may suggest a role for CaBP7 in the regulation of Ca2+ signalling processes at the lysosomal membrane during both interphase and in dividing cells. Finally the use of nuclear magnetic resonance (NMR) spectroscopy led to the partial backbone and side chain assignment of a 100 amino acid N-terminal fragment of CaBP7. Using this data the secondary structure of this fragment was calculated and closely resembled that of Ca2+-bound CaM and CaBP1S N-termini. Spectra for apo CaBP7 were distinct from that of Ca2+-saturated CaBP7 implying that the protein undergoes a significant conformational change upon Ca2+ binding. In addition, the use of native PAGE demonstrated a shift in mobility of CaBP7 1-100 upon addition of Ca2+ but not Mg2+. Continued NMR analysis of CaBP7 will be required for the determination of a high-resolution structure for this protein.
|Item Type:||Thesis (Doctoral)|
|Subjects:||Q Science > QP Physiology|
|Departments, Research Centres and Related Units:||Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Biomedical Sciences|
|Deposited On:||07 Aug 2012 09:17|
|Last Modified:||25 Oct 2014 01:00|
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