ASAP is committed to accelerating the pace of discovery and informing a path to a cure for Parkinson’s disease through collaboration, research-enabling resources, and data sharing. We’ve created this catalog to showcase the research outputs and tools developed by ASAP-funded programs.
Protein purification protocol for tag-less LRRK2RCKW as done by Leschziner and Reck-Peterson Labs. Same protocol can be used to purify LRRK1RCKW as well. Original protocol by David Snead. Modified by Yu Xuan Lin and Mariusz Matyszewski for publication.
PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074, and Thr1075) within the CORB GTPase domain
Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause PD. Here, the authors study the mechanism controlling LRRK1 activity and reveal a novel unexpected activation mechanism. View preprint.
Published: PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074, and Thr1075) within the CORB GTPase domain
Published: This study describes how PKC isoforms are able to phosphorylate LRRK1 at three sites in a key regulatory domain of the protein (GTPase domain) inducing LRRK1’s kinase activity. Interestingly, this is not seen with the PD-associated LRRK2, suggesting that PKC isoforms do not regulate LRRK2. View original preprint.
Protocol for expressing LRRK1 and LRRK2 in insect cells.
This assay uses TMR labeled LRRK2 or LRRK1 RCKW to measure binding to microtubules in vitro
A mono- and intralink filter (mi-filter) to reduce false identifications in cross-linking mass spectrometry data
The authors show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.
This protocol assumes that 2000 to 4000 movies of full-length LRRK2 embedded on vitreous ice are collected with an electron microscope equipped with a direct detector. It focuses primarily on the monomeric population of LRRK2 and has been leading to 3-4 Å resolution structures.
This quick guide provides key minimal steps for preparing the Titan/SerialEM for the tomogram data collection on lamella or in vitro specimens with a K3 camera. paceTOMO routine is also included for a typical tomogram data collection session. Please note that this is not an exhaustive guide, but summarizes the order of key steps.
Review: This review focuses on new insights into the stucture of LRRK2’s cytosolic and microtubule-bound forms and challenges going forward.
Preprint: LRRK2 mutations are a common cause of familial PD. In some circumstances, LRRK2 co-localizes with microtubules. The authors report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase (closed conformation) and GTPase domains, bound to microtubules. Further, they identified amino acids that mediate microtubule binding.