Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly

摘要: Fetal or adult rat-brain cytosol and fetal microtubules contain a high-affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30–50 nM range. best competitors (in decreasing order) are pregnenolone sulfate, progesterone, Δ5-pregnene-3β,20α-diol, 3β-hydroxy-5α-pregnan-20-one. It was hypothesized that protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of brain by fast pressure liquid chromatography with sequential ion-exchange gel-filtration columns yielded two fractions, one very high molecular mass, >200 kDa, other 40–60 enriched [3H]pregnenolone-binding activity immunolabeled monoclonal anti-tubulin anti-MAP2 antibodies. Because many associated microtubules, binding assays were repeated purified calf-brain tubulin, MAP2, Tau Only MAP2 fraction showed saturable [3H]pregnenolone an affinity close but much larger concentration sites (16 pmol/mg MAP2), which increased more than 8-fold after copolymerization tubulin. Finally, steroid effects on microtubule-assembly kinetics assayed. Pregnenolone induced large, dose-related increase both rate extent MAP2-induced tubulin assembly, whereas inactive per se, counteracted stimulatory effect pregnenolone. Electron microscopic analysis confirmed pregnenolone-increased assembly produced completely normal structure. MAP2–tubulin interaction also observed neuron cultures. Therefore, we propose mechanism neurosteroid action, control microtubule or, generally, neural cytoskeleton dynamics, potential roles development, plasticity, aging.