Biochemical characterization of the cAMP-dependent protein kinase regulatory subunit-like protein from Trypanosoma equiperdum, detection of its inhibitory activity, and identification of potential interacting proteins.
An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5-20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T. equiperdum PKA regulatory subunit-like protein (TeqR-like) on the basis of Western blot and mass spectrometry analyses, and behaved as an uncompetitive or anti-competitive inhibitor of the parasite TeqC-like protein, with respect to a fluorescently labeled substrate (kemptide, sequence: LRRASLG), showing a Ki of 1.17 μM. The isolated protein possesses a molecular mass of 57.54 kDa, a Stokes radius of 3.64 nm, and a slightly asymmetric shape with a frictional ratio f/fo = 1.43. As revealed during the purification steps and by immunoprecipitation experiments, the TeqR-like and TeqC-like proteins were not associated forming a heterooligomeric complex, differing from traditional PKA subunits. Co-immunoprecipitation results followed by mass spectrometry sequencing identified two isoforms of the parasite heat-shock protein 70, α-tubulin, and β-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum.
['Araujo|Nelson A|NA|', 'Rincón|Mónica|M|', 'Vonasek|Eva|E|', 'Calabokis|Maritza|M|', 'Bubis|José|J|']
D000818:Animals D017868:Cyclic AMP-Dependent Protein Kinases D008024:Ligands D021122:Protein Subunits D013552:Swine D014345:Trypanosoma
Biochemical characterization Trypanosoma equiperdum Dourine Protein purification Protein-protein interactions Regulatory and catalytic subunits of the cAMP-dependent protein kinase Trypanosomatid parasites Uncompetitive or anti-competitive inhibition