Open Collections

Characterization of hippocalcin-like protein 1 (HPCAL1), a neuronal calcium sensor protein in the retina

University of British Columbia

Hippocalcin-like protein 1 (HPCAL1) is a neuronal calcium sensor (NCS) protein found in the brain and in the retina. NCS proteins have important roles in signaling pathways including phototransduction; however, the role of HPCAL1 in the retina remains unresolved. The objective of this thesis is to characterize HPCAL1 and identify its potential interacting partners in the retina. The first part of the thesis examines the localization and characteristics of HPCAL1 using a variety of biochemical assays. The presence of HPCAL1 in the retina was confirmed with RT-PCR, Western blotting analysis, and immunofluorescence microscopy. Since NCS proteins respond to intracellular calcium level changes, HPCAL1 was expressed and isolated from both mammalian cells and E. coli to study its calcium binding properties and other characteristics. Results from a gel mobility shift assay and a fluorescence assay clearly indicated that HPCAL1 undergoes conformational changes upon calcium binding. Furthermore, membrane association assays confirmed that retinal HPCAL1 possesses the calcium-myristoyl switch mechanism which responds to the presence or absence of calcium. NCS proteins often interact with other proteins to perform their functions; therefore, the second part of the study involves the use of mass spectrometry in an attempt to identify calcium-dependent interacting partners of HPCAL1. Over 300 potential interacting partners were identified, and selected proteins were subjected to co-immunoprecipitation and Western blotting analysis or co-localization studies using immunofluorescence microscopy in order to confirm their interactions with HPCAL1. TorsinA has been identified as a calcium-dependent interacting partner to HPCAL1; however, further studies will have to be conducted to determine the significance of this interaction and to confirm other potential interacting partners that were identified in the mass spectrometry analysis. The results of this study provide important technical information on the biochemical characterization of HPCAL1 and the properties of HPCAL1 in the retina. Not only has the identification of potential interacting partners of HPCAL1 provided first insights at its possible role or function in the retina, it has also pointed out the potential for identifying interacting partners of other NCS proteins using mass spectrometry.

Text

2015-08-20

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/54575

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/