Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations.
Previously, we have demonstrated the presence of a protein factor [tubulin polymerization perturbing protein (TPPP)] in brain and neuroblastoma cell but not in muscle extract that uniquely influences the microtubule assembly. Here we describe a procedure for isolation of this protein from the cytosolic fraction of bovine brain and present evidence that this protein is a target of both tubulin and microtubules in vitro. The crucial step of the purification is the cationic exchange chromatography; the bound TPPP is eluted at high salt concentrations, indicating the basic character of the protein. By IDA-nanoLC-MS analysis of the peptides extracted from the gel-digested purified TPPP, we show the presence of a single protein in the purified fraction that corresponds to p25, a brain-specific protein the function of which has not been identified. Circular dichroism data have revealed that, on one hand, the alpha-helix content of p25 is very low (4%) with respect to the predicted values (30-43%), and its binding to tubulin induces remarkable alteration in the secondary structure of the protein(s). As shown by turbidimetry, pelleting experiments, and electron microscopy, p25 binds to paclitaxel-stabilized microtubules and bundles them. p25 induces formation of unusual (mainly double-walled) microtubules from tubulin in the absence of paclitaxel. The amount of aberrant tubules formed depends on the p25 concentration, and the process occurs at substoichiometric concentrations. Our in vitro data suggest that p25 could act as a unique MAP in vivo.
Hlavanda
Emma
E
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, POB 7, H-1518 Hungary.
Kovács
János
J
Oláh
Judit
J
Orosz
Ferenc
F
Medzihradszky
Katalin F
KF
Ovádi
Judit
J
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism* / Q000473:pathology* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D050257:Tubulin Modulators
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D014404:Tubulin; D050257:Tubulin Modulators