Biochemical characterization of full length human recombinant LRRK1 and LRRK2

Promising Outcomes of Original Grant:
In our initial application we proposed to take advantage of a lentiviral system recently developed in KULeuven to express full-length, soluble human LRRK1 and LRRK2 to allow a thorough biochemical characterization of the two proteins. We felt that an efficient system to purify LRRK2 was missing, along with a side-by-side characterization of the homologous protein LRRK1, which is not linked to Parkinson’s disease. Thanks to the Fox Foundation that funded this grant and to the collaborative spirit of the project that takes advantage of expertise from three laboratories, we have accomplished most of the goals and we are now looking forward to using both cell lines and recombinant proteins to further explore the “social” role of LRRK2 as an intracellular signaling molecule.

Objectives for Supplemental Investigation: 
We are very interested in finding LRRK2 interacting proteins as this may provide important clues on LRRK2 biological function along with the possibility of modifying LRRK2 activity by acting on other components of the signaling pathway. In particular, we will search for those interactors that are specific for the activated state of LRRK2, which depends on the binding of the small nucleotide GTP to a specific region of the protein, the ROC domain. To do so, we will use two independent techniques. One based on a high throughput screening of about 10,000 synthetic proteins spotted on a micro-array glass that will be incubated with LRRKs recombinant proteins in the presence or absence of GTP. The second strategy is based on co-immunoprecipitation coupled with mass-spectrometry using our stable, low-expressing cell lines to capture LRRK2 interactors.

Importance of This Research for the Development of a New PD Therapy: 
Identification of LRRK2 interactors, especially those specific for the GTP-bound state of the protein, is pivotal in the sense of therapeutic implication. If we know precisely the “upstream” molecules that regulate LRRK2 activity, we could block their function to indirectly stop LRRK2 activity. For instance, there are a number of pharmacological compounds blocking “upstream” signaling proteins already approved by FDA to treat a number of cancers. Furthermore, knowing the molecules that directly modulate LRRK2 activity is also crucial for the development of therapeutic approaches. Finally, the identification of LRRK2 “downstream” effectors may be extremely useful for diagnosis as the signal is amplified from the top to the bottom of the cascade and, therefore, while we might not appreciate LRRK2 activation we could be able instead to measure activation of its downstream targets.