Unit:
Διατμηματικό / Διϊδρυτικό ΠΜΣ Μοριακή ΙατρικήLibrary of the School of Health Sciences
Author:
Λεάνδρου Εμμανουέλα
Supervisors info:
Λεωνίδας Στεφανής, Κώστας Βεκρέλλης, Σπύρος Ευθυμιόπουλος
Original Title:
Novel approaches to purify and measure the activity of dimeric and monomeric LRRK2
Translated title:
Απομόνωση και μέτρηση της ενεργότητας μονομερούς και διμερούς LRRK2
Summary:
The ROCO/RIP kinase protein LRRK2, mutations in which are causally linked to
Parkinson’s disease (PD), exists in a broad range of monomeric to highly
oligomeric species regulated by a multitude of factors. Until now, all the
methods used to detect oligomerization of LRRK2 were based on the molecular
mass of the protein (size-exclusion chromatography, native blue-PAGE) or on in
vitro detection of oligomeric LRRK2 through co-immunoprecipitation experiments.
However, these methods cannot discriminate between truly oligomeric species of
LRRK2 or monomeric species of LRRK2 bound to a protein complex. In this study,
we combined a proximity biotinylation assay and an ELISA-based kinase assay, in
order to purify and measure the kinase activity of the different conformation
states of LRRK2. The proximity biotinylation assay permits the detection and
purification of LRRK2 dimers or oligomers after their formation in living
cells. Thereby, the oligomers are formed inside their natural environment where
the physiology of the protein is not perturbed. The toxicity induced by LRRK2
pathogenic mutations has been linked with altered kinase activity. Detection of
differences in the kinase activity between the different conformation states of
LRRK2 is of great importance. If the conformation state of the protein plays a
role in the kinase activity, then specific inhibitors can be produced that will
inhibit only the activity of the conformation responsible for increased kinase
activity. Thus, a possible pathogenic kinase activity can be inhibited without
affecting the steady state levels of activity that might be crucial for cell
survival.
Keywords:
Parkinson's Disease, LRRK2 protein, Oligomerization, Kinase activity, Proximity biotinylation assay
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