The LRRK2 protein is regulated by modifications called phosphorylation, which occurs at several specific sites on the protein (Serines910, 935, 955, 973) and can alter the biology of LRRK2. When LRRK2 activity is blocked by small molecule inhibitors, or in some Parkinson's disease (PD)-associated LRRK2 mutants, the phosphorylation of these sites is disrupted. We found that this causes the addition of another kind of modification called ubiquitination, which can alter the stability or function of LRRK2. The link between PD mutations and small molecule inhibition, a potential therapeutic avenue for PD, indicates an important regulatory mechanism for LRRK2. This work will create a measurable system to detect the phosphorylation/ubiquitination of LRRK2 in cells. This will provide a platform to understand this regulatory cycle of LRRK2 modification.
Cellular systems are ideal to study the molecular players in a regulatory scheme because they are open to genetic and pharmacological changes from new potential targets. We will establish tools to detect variations in LRRK2 modifications that can be adapted to a multi-well format to enable testing of multiple regulators of LRRK2 phosphorylation at once.
We will first establish a system to detect the addition of ubiquitin molecules to LRRK2 with a method that relies on the joining of ubiquitin to LRRK2 to generate a signal. We will develop and test these assays in cells where we have introduced extra LRRK2. It also will be important to perform our tests in cells with normal levels of LRRK2. To do this, we will use gene editing techniques to attach DNA sequence encoding to a molecular handle (or protein tag) at the beginning of the LRRK2 gene. This handle will be used in the ubiquitination detection assay. We will combine these new cell lines and the ubiquitin detection assay to measure the change in ubiquitin on LRRK2 in cells under various conditions.
Impact on Diagnosis/Treatment of Parkinson's disease:
The phosphorylation of LRRK2 is linked to its pathology through modulation by LRRK2 inhibitors and disease-associated mutations. Inhibition induces dephosphorylation and subsequent ubiquitination of LRRK2, which has on-target molecular implications for a potential therapeutic avenue for PD. Our proposal will provide a means to understand this process and identify the ubiquitin regulators of LRRK2.
Next Steps for Development:
The assays and cell line tools developed and employed in this proposal will allow us to screen for the LRRK2 ubiquitin modifiers. This LRRK2-ubiqutination detection assay will next be used to identify the enzymes that modify the LRRK2 ubiquitination using libraries of drugs or genetic tools that inhibit or enhance the activity of these ubiquitin modifiers.