The initiative for our research in this field was the discovery of a new tubulin-targeting protein. We noticed that an extract from brain, but not from muscle, contains a protein that promotes formation of tubulin assemblies. The purified protein from bovine brain was identified as the basic, heat resistant, about 25 kDa protein that had been found in a partially purified fraction of bovine tau protein kinase II. The bovine and human proteins (termed p25) were cloned, and found to exhibit 90% structural identity.

The same protein was also described as p24, a glycogen synthasekinase-3 inhibitor, and as p25alpha, an alpha-synuclein bindingprotein. To make clear that p25, p24 and p25alpha are the same protein(NCBI accession #2498194) that map to the p15.3 region of humanchromosome 5, and entirely different from the truncated form (p25) ofthe regulatory subunit, p35, of cyclin-dependent kinase-5, we named the newly purified protein to TPPP/p25, Tubulin Polymerization Promoting Protein. The amino acid sequence of this protein differs from that ofother proteins significantly identified so far, but it shows high homology with two hypothetical proteins detected via BLAST. Thus TPPP/p25is the first member of a new protein family. An unexpected finding was the discrepancy between the predicted and the demonstrated secondary structure elements of TPPP/p25. Nuclear magneticresonance and fluorescence data revealed that TPPP/p25 does not have well-defined structure and it belongs to the group of intrinsically unstructured proteins even if it shows flexible structural elements. These results agree with the in silico structural prediction, namely, that the N-terminal region of TPPP/p25 is unfolded and is followed by a segment (40 residues long) of ordered structure. This ordered segmentmay contain the beta-structure. TPPP/p25 is considered to be a phosphoprotein but the function of the post-translation modification is still unclear. A characteristic feature of unstructured proteins is formation of protein-protein interactions that are promoted by the crowded intracellular milieu, thus failing their destined degradation by the proteolytic machinery. The major target of TPPP/p25 is the tubulin/MT system in vitro and in living cell. In vitro it induces the formationof intact-like MTs, double walled tubules and polymorphic aggregates at substoichiometric concentration, and bundles paclitaxel-stabilized MTs as judged by electron and atomic force microscopies. In living eukaryotic cells (HeLa, NRK and SK-N-MC) at low expression level TPPP/p25 co-localizes specifically with the microtubular system without affecting the cell cycle process (cf. figure). This is possible since the co-localization (heterologous association) is dynamic (t1/2 = 5 sec by fluorescence recovery after photobleaching analysis), and it is changed during the mitotic phases. The bundled MTs by TPPP/p25 observedin living cells were resistant to the destabilizing effect of vinblastine. Thus TPPP/p25 seems to behave as a MAP, like tau protein, which has some characteristics in common with TPPP/p25. This suggests that a potential biological function of TPPP/p25 is to contribute to maintain the optimal balance between the mechanical rigidity of MTs and their polymerization dynamics. The TPPP/p25 gene was reported to be expressed in humans as mRNA in distinct brain areas mainly. Immunohistochemical studies showed TPPP/p25 protein is expressed in oligodendrocytes of human brain tissue. TPPP/p25inclusions characteristic for a-synucleinopathies (e.g. Parkinson's disease) but not for tauopathies (e.g. Alzheimer disease) were identified in human pathological brain tissues. The enrichment of TPPP/p25 in inclusions of neuronal and glia cells of human brain tissues has been demonstrated by immunohistochemistry. In the case of multiple system atrophy complete and partial co-localization with a-synuclein and tubulin, respectively, was revealed. The inclusion ofTPPP/p25 in large protein aggregates could be mimicked at the cellular level by over-expression of TPPP/p25 in HeLa cells. Formation of a large protein aggregate involving TPPP/p25 is promoted by inhibition of the proteosome machinery. Impaired degradation of this unstructured protein may lead to itsassociation/aggregation with a-synuclein, tubulin or other not yet identified proteins. The formation of such a superstructure(aggresome/inclusion body) results finally in cell death. Whether the TPPP/p25-containing superstructure ensures a cytoprotecting mechanism (hereby impeding cell death) against the toxic effect of the accumulated unstructured protein, is still unrevealed.