Identification of peptides interfering with the LRRK2/PP1 interaction

Abstract

Serine/threonine phosphatases are responsible for modulating the activities of the protein kinases implicated in the development of several pathologies. Here we identified by a PEP-scan approach a peptide of LRRK2, a Parkinson’s disease associated protein, interacting with the phosphatase PP1. In order to study its biological activity, the peptide was fused via its N-terminal to an optimized cell penetrating peptide. We synthesized from the original peptide five interfering peptides and identified two (Mut3DPT-LRRK2-Short and Mut3DPT-LRRK2-Long) able to disrupt the LRRK2/PP1 interaction by competition in anti-LRRK2 immunoprecipitates. Using FITC-labelled peptides, we confirmed their internalization into cell lines as well as into primary cells obtained from healthy or ill human donors. We confirmed by ELISA test the association of Mut3DPT-LRRK2-Long peptide to purified PP1 protein. The peptides Mut3DPT-LRRK2-5 to 8 with either N or C-terminal deletions were not able to disrupt the association LRRK2/PP1 nor to associate with purified PP1 protein. The interfering sequences blocking the PP1/LRRK2 interaction were also fused to a shuttle peptide able to cross the blood brain barrier and showed that the newly generated peptides BBB-LRRK2-Short and BBB-LRRK2-Long were highly resistant to protease degradation. Furthermore, they blocked PP1/LRRK2 interaction and they penetrated into cells. Hence, these newly generated peptides can be employed as new tools in the investigation of the role of the LRRK2/PP1 interaction in normal and pathological conditions.

PEPscan: A Broad Spectrum Approach for the Characterization of Protein-Binder Interactions?

“…For this, we consider the identification of PP1a sequences involved in binding to one FITC-labeled LRRK2 peptide. We have previously identified and published the sequence of the interfering peptide, blocking the interaction between the kinase LRRK2 and the phosphatase PP1 [ 13 ]. As LRRK2 was not available commercially at the time of the study, it was not possible to use the same protocol to identify the peptide of PP1, blocking the interaction with LRRK2.…”

“…More recently, PEPscan has gained interest for the modulation of protein-protein interactions, with several reports showing its effectiveness to identify peptides able to interfere with protein-protein interactions [ 12 ]. A very recent similar approach concerns the interference between LRRK2 and PP1 in the context of Parkinson’s disease [ 13 ]. In these experiments, one hypothesizes that the active peptides identified are located at the protein-protein interface, but in fact very little is known about the exact location of those peptides, relative to the binding interface, as PEPscan is usually employed in cases where the structure of the complex involving the proteins of interest is not known (see [ 14 ]).…”

Pepscan Approach for the Identification of Protein–Protein Interfaces: Lessons from Experiment

“…Other PEPscan experiments for which no experimental 3D structure could be identified were not included in this data set. This is the case of the experiment targeting the interaction between two proteins of the parasite Plasmodium falciparum , the phosphatase PP1 and the protein LRR1, involved in malaria parasite development [ 43 ], and of the recently published study targeting the phosphatase PP1 and the kinase LRRK2 involved in Parkinson disease [ 44 ] (Patent PCT 20141031).…”

Leucine rich repeat kinase 2 (LRRK2) peptide modulators: Recent advances and future directions

“…[ 89 ] Recently, cell penetrating peptides were designed to effectively disrupt the interaction between LRRK2 and protein phosphatase 1 (PP1), which dephosphorylates LRRK2. [ 90 ] Initial work determined that these peptides were able to permeate cells, resist proteolytic degradation, and block the interaction between LRRK2 and PP1. These peptides can be utilized to better understand how this PPI drives LRRK2 pathogenicity, but there are multiple PPIs involving LRRK2 that are known to directly regulate LRRK2 activity.…”