Artículo de revista
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
Fecha
2020Registro en:
Biochimie 168 (2020) 110-123
10.1016/j.biochi.2019.10.020
Autor
Araujo, Nelson A.
Rincón, Mónica
Vonasek, Eva
Calabokis, Maritza
Bubis, José
Institución
Resumen
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 K-i of 1.17 mu M. 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, alpha-tubulin, and beta-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum.
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