Comparative Analysis of Different Peptidyl-Prolyl Isomerases Reveals FK506-binding Protein 12 as the Most Potent Enhancer of α-Synuclein Aggregation

Angélique Deleersnijder,Anne-Sophie Van Rompuy,Linda Desender,Hans Pottel,Luc Buée,Zeger Debyser,Veerle Baekelandt,Melanie Gerard

doi:10.1074/jbc.m110.182303

Abstract

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.

Highlights

Parkinson disease (PD)2 is the second most common neurodegenerative disorder
We demonstrated that FKBP12 and FKBP52 enhance the aggregation of ␣-SYN in a neuronal cell culture model for synucleinopathy [15]
We have previously demonstrated that FKBP12 and FKBP52 stimulate the aggregation of ␣-SYN in vitro and/or in live cells [14, 15]

Summary

Introduction

Parkinson disease (PD)2 is the second most common neurodegenerative disorder. Accumulating evidence points to a caus-. Using two different selection markers allowed us to make double overexpression SHSY5Y cell lines (␣-SYN and CYPA, Pin1, FKBP12, FKBP38, FKBP52, or FKBP65). Enzymatic Characterization of Different PPIases—To study the effect of FKBP52, FKBP65, Pin1, and CYPA on the aggregate and fibril formation of ␣-SYN in vitro, recombinant proteins were produced and purified (see “Experimental Procedures” and supplemental Fig. S1).

Results

Conclusion