Project overview
This project is focused on developing an experimental approach and new
avenue of research associated with understanding important disease mechanisms. In particular, in order to delineate the functional mechanisms of LRRK2’s role in cells, we will take advantage of a cutting-edge technique to label in situ adjoining proteins that make up key protein complexes, via fusion of a proximity-dependent ascorbate peroxidase called APEX2 onto our gene of interest, LRRK2. APEX2 is an enzyme that catalyses the oxidation of biotin-phenol, eventually resulting in the covalent attachment of biotin to other proteins that lie within a nanometer range. The advantage of using APEX2 is that
neighbouring proteins can be labelled quickly, within minutes, and therefore acute changes in protein complex formation can be monitored. Following biotin labelling, these interacting proteins can then be identified biochemically via Western blot or mass spectrometry following Streptavidin pulldown from cell lysates. Therefore, this technique can identify discrete protein complex constituents within a physiological setting, under strict control and following an acute stimulus, not attainable with any other system. Use of this technique to understand LRRK2 function and its relationship to mitochondrial health is an extremely relevant area implicated in disease pathology and an important area for study within the research community, which links well with our ongoing projects understanding mitochondrial quality control mechanisms. Therefore, this experimental system will be powerful in opening new areas of research within my laboratory and will be important to establish preliminary data for future grant proposals to understand the fundamental mechanisms driving
neurodegenerative disorders.
avenue of research associated with understanding important disease mechanisms. In particular, in order to delineate the functional mechanisms of LRRK2’s role in cells, we will take advantage of a cutting-edge technique to label in situ adjoining proteins that make up key protein complexes, via fusion of a proximity-dependent ascorbate peroxidase called APEX2 onto our gene of interest, LRRK2. APEX2 is an enzyme that catalyses the oxidation of biotin-phenol, eventually resulting in the covalent attachment of biotin to other proteins that lie within a nanometer range. The advantage of using APEX2 is that
neighbouring proteins can be labelled quickly, within minutes, and therefore acute changes in protein complex formation can be monitored. Following biotin labelling, these interacting proteins can then be identified biochemically via Western blot or mass spectrometry following Streptavidin pulldown from cell lysates. Therefore, this technique can identify discrete protein complex constituents within a physiological setting, under strict control and following an acute stimulus, not attainable with any other system. Use of this technique to understand LRRK2 function and its relationship to mitochondrial health is an extremely relevant area implicated in disease pathology and an important area for study within the research community, which links well with our ongoing projects understanding mitochondrial quality control mechanisms. Therefore, this experimental system will be powerful in opening new areas of research within my laboratory and will be important to establish preliminary data for future grant proposals to understand the fundamental mechanisms driving
neurodegenerative disorders.
