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PMID:19022250

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Citation

Levin, J, Giese, A, Boetzel, K, Israel, L, Högen, T, Nübling, G, Kretzschmar, H and Lorenzl, S (2009) Increased alpha-synuclein aggregation following limited cleavage by certain matrix metalloproteinases. Exp. Neurol. 215:201-8

Abstract

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.

Links

PubMed Online version:10.1016/j.expneurol.2008.10.010

Keywords

Biophysical Processes; Matrix Metalloproteinase 1/pharmacology; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases/pharmacology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods; Spectrum Analysis; alpha-Synuclein/drug effects; alpha-Synuclein/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:MMP1

involved_in

GO:0031334: positive regulation of protein-containing complex assembly

ECO:0000314: direct assay evidence used in manual assertion

P

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP1

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP1

involved_in

GO:0006508: proteolysis

ECO:0000314: direct assay evidence used in manual assertion

P

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP3

involved_in

GO:0031334: positive regulation of protein-containing complex assembly

ECO:0000314: direct assay evidence used in manual assertion

P

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP3

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP3

involved_in

GO:0006508: proteolysis

ECO:0000314: direct assay evidence used in manual assertion

P

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP9

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP9

involved_in

GO:0006508: proteolysis

ECO:0000314: direct assay evidence used in manual assertion

P

  • has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP9

involved_in

GO:0006508: proteolysis

ECO:0000314: direct assay evidence used in manual assertion

P

has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP9

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

has_direct_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP1

involved_in

GO:0006508: proteolysis

ECO:0000314: direct assay evidence used in manual assertion

P

has_input:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP1

involved_in

GO:0032461: positive regulation of protein oligomerization

ECO:0000314: direct assay evidence used in manual assertion

P

has_regulation_target:(UniProtKB:P37840)

Seeded From UniProt

complete

HUMAN:MMP1

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

has_direct_input:(UniProtKB:P37840)

Seeded From UniProt

complete

Notes

See also

References

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