Abstract

TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.

Highlights

  • TPPP/p25 (Tubulin Polymerization Promoting Protein) is a brain specific protein which binds to tubulin and induces MT bundle formation both in vitro and in cells[12,13]

  • The only biological function attributed to native TPPP/p25 is related to its ability to bind and bundle MTs, offering an explanation as to how TPPP/p25 may influence the plasticity of MT networks in neuronal cells

  • Both N- and C-terminal regions are intrinsically disordered and highly basic and one can suggest that the binding of both domains to microtubules is achieved through electrostatic interactions with the acidic C-terminal tails of tubulin extending from the MTs (Fig. S1B)

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Summary

Introduction

TPPP/p25 (Tubulin Polymerization Promoting Protein) is a brain specific protein which binds to tubulin and induces MT bundle formation both in vitro and in cells[12,13]. TPPP/p25 co-localizes selectively with the microtubule network in eukaryotic cells causing stabilization of the system; the overexpression of this protein in transfected HeLa cells induces a characteristic protein aggregation reminiscent of the process of aggresome formation[26]. This process may be related to the enrichment of TPPP/p25 in inclusion bodies in the brains of patients afflicted with Parkinson’s disease or other synucleinopathies[18,19,27]. Using Bimolecular fluorescence complementation assays in cells[31], we demonstrate that the bundling activity of TPPP/p25 is achieved by homotypic protein-protein interactions mediated by the central p25 domain depending on the ability of the protein to bind MTs

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