Increased alpha-synuclein aggregation following limited cleavage by certain matrix metalloproteinases.
Recent evidence indicates that protein aggregation and in particular the formation of toxic protein oligomers is a key mechanism in synucleinopathies such as Parkinson's disease (PD). Post mortem brain tissue studies as well as animal studies furthermore suggest that matrix metalloproteinases (MMPs) are also involved in the pathogenesis of PD. We used confocal single molecule spectroscopy to characterize the influence of MMPs and other proteases on the aggregation of alpha-synuclein. These studies were complemented by the characterization of alpha-synuclein fragment patterns generated by these proteases using gel electrophoresis and mass spectrometry. Limited digestion by MMP-1 and MMP-3, but not by MMP-9, increased the tendency of alpha-synuclein to aggregate. Proteinase K and Trypsin did not increase the level of de novo aggregation of alpha-synuclein. SDS-PAGE as well as MALDI-ToF analysis of limitedly digested alpha-synuclein demonstrate that all proteases generate different fragments of alpha-synuclein. We provide mass spectrometry data of proteolytic alpha-synuclein fragments and propose specific cleavage sites for MMP-1 and MMP-9 in alpha-synuclein. We furthermore found four additional cleavage sites of MMP-3 that had not been described previously. In order to increase aggregation of alpha-synuclein, specific cleavage between the highly charged C-terminal domain and the aggregation-prone NAC domain of alpha-synuclein seems to be crucial. Our findings obtained in vitro in a well-characterized model of pathological alpha-synuclein aggregation indicate that MMP-1 and MMP-3 may also influence pathogenesis of PD in vivo by generation of specific aggregation-enhancing alpha-synuclein fragments resulting from limited proteolysis.
Levin
Johannes
J
Neurologische Klinik, Klinikum Grosshadern, Ludwig-Maximilians-Universität München, Germany. jlevin@med.uni-muenchen.de
Giese
Armin
A
Boetzel
Kai
K
Israel
Lars
L
Högen
Tobias
T
Nübling
Georg
G
Kretzschmar
Hans
H
Lorenzl
Stefan
S
D055592:Biophysical Phenomena; D020781:Matrix Metalloproteinase 1 / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D020782:Matrix Metalloproteinases / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013057:Spectrum Analysis; D051844:alpha-Synuclein / Q000187:drug effects* / Q000378:metabolism*
D051844:alpha-Synuclein; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1