Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

J Vis Exp. 2017 Aug 13;(126):55361. doi: 10.3791/55361.

Abstract

α-Synuclein (aSyn), β-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo.

Publication types

  • Video-Audio Media

MeSH terms

  • Catalytic Domain
  • Cell-Free System
  • Colorimetry / methods*
  • Humans
  • Protein Phosphatase 2 / metabolism*
  • Recombinant Proteins / metabolism*
  • alpha-Synuclein / genetics
  • alpha-Synuclein / metabolism
  • beta-Synuclein / metabolism
  • gamma-Synuclein / genetics
  • gamma-Synuclein / metabolism*

Substances

  • Recombinant Proteins
  • alpha-Synuclein
  • beta-Synuclein
  • gamma-Synuclein
  • Protein Phosphatase 2