Allosteric inhibition of LRRK2 with Rab mimitics

de Jong, Brecht (2019) Allosteric inhibition of LRRK2 with Rab mimitics. Bachelor's Thesis, Chemistry.

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Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by tremor. This tremor often starts in the hands but gradually spreads and worsens. Currently it cannot be cured. Leucine-rich repeat kinase 2 (LRRK2) is a kinase that plays an important role in PD. It can increase the risk or even be causative for PD. In PD the kinase activity of LRRK2 is increased. Naturally the kinase activity of LRRK2 increases by being recruited to the trans-Golgi membrane where it becomes a dimer. As a dimer on the trans-Golgi membrane it is more active than as a monomer in the cytosol. We want to use stapled peptides to inhibit the recruitment to the trans-Golgi membrane so that LRRK2 activity will be lowered. It was found that the stapled peptides we used bind to GFP-LRRK2. These stapled peptides did not show an effect on LRRK2 localization in HEK293 cells transfected with GFP-LRRK2. Repeating the experiment in A549 cells with endogenous LRRK2 was considered but ruled out later. The pulldown of endogenous LRRK2 did not show binding of the stapled peptides. A kinase assay was done twice in RAW264.7 cells to study the effect of stapled peptides on the kinase activity. The first kinase assay showed a clear decrease in kinase activity upon adding stapled peptides for each stapled peptide. The second kinase assay only shows this effect for stapled peptide Rab29III. It must be noted that as a control MLi2 was added which is a kinase inhibitor but in the first assay it did not show a decrease in the kinase activity but in the second assay it did. Overall it is promising that the stapled peptides bind to GFP-LRRK2 but the localization did not give the expected result. The kinase assays seems to indicate that stapled peptides do reduce the kinase activity. In the future it would be recommended to do the localization experiments for Rab32 as well and to label the lysosomes with LAMP1. Further investigation on the kinase assay would also be valuable so that better results can be obtained.

Item Type:	Thesis (Bachelor's Thesis)
Supervisor name:	Kortholt, A.
Degree programme:	Chemistry
Thesis type:	Bachelor's Thesis
Language:	English
Date Deposited:	12 Jul 2019
Last Modified:	12 Jul 2019 11:56
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/20145

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