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

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.

Alternative Structures of α-Synuclein

“…Co-immunoprecipitation is also considered as a potential therapeutic technique [46]. Selection of the 30-100 fragment based on analysis of the ASyn amyloid structure remains consistent with the outcome of experimental studies which single out the NAC fragment (60-100) as particularly prone to amyloid transformation [47] An open question is why the fragments at 1-30 and 100-140, despite repeating the same sequence in all unit chains, do not participate in propagation of the fibril.…”

Sensitization of FOLFOX-resistant colorectal cancer cells via the modulation of a novel pathway involving protein phosphatase 2A

“…We determined PP2A activity by using recombinant human PP2A-Cα protein and threonine phosphopeptide (KRpTIRR) substrate following the Malachite green protocol as previously described (Lek et al, 2017). The exact protocol we followed for the PP2A assay is given at: https://www.jove.com/pdf/55361/jove-protocol-55361-recombinant-synucleins-stimulateprotein-phosphatase-2a-catalytic.…”

“…We also determined the effect of NSC49L treatment on the stimulation of PP2A-Cα in FOLFOX-HT29 cells. The immunocomplexes from the control and treated groups were isolated with anti-PP2A-C antibody and were used for PP2A-Cα activity (Lek et al, 2017). Results showed an increased PP2A activity in a dose-dependent manner (Fig.…”

“…First, we performed an in vitro PP2A activity assay using recombinant PP2A-Cα protein and threonine phosphopeptide (KRpTIRR) as substrate in a Malachite green assay (Lek et al, 2017). NSC49L stimulated PP2A-Cα activity in a dose-dependent manner with a Kact(NSC49L) of 14 nM and Vmax of 3384 nmoles/mg/min (Fig.…”

Sphingosine‐1‐phosphate receptor‐independent lung endothelial cell barrier disruption induced by FTY720 regioisomers

“…Reactions proceeded 10 min at 30 C with intermittent shaking in the presence of pT substrate described above. Reactions were stopped by placing on ice, with aliquots evaluated by malachite green assay relative to freshly prepared phosphate standards and read at 630 nm (Multiskan Spectrum plate reader, Thermo Scientific, Pittsburgh, PA) as detailed in Lek et al 31 PP2A activity is expressed as pmol phosphate/min/mg protein normalized to the methanol vehicle as baseline.…”