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Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling.

DeBonis S, Neumann E, Skoufias DA - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Université de Grenoble Alpes, F-38044 Grenoble, France.

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.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the possible interaction modes of TPPP/p25 resulting in microtubule binding and bundling.The various elements and distances are not drawn to scale.
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f8: Schematic representation of the possible interaction modes of TPPP/p25 resulting in microtubule binding and bundling.The various elements and distances are not drawn to scale.

Mentions: The presence of two MT binding sites on TPPP/p25 provides an obvious model for how one molecule of TPPP/p25 can crosslink two MTs, with each binding site interacting with a different MT (Fig. 8, models A and C). Alternatively, one of the sites on TPPP/p25 might bind a tubulin site which is masked when tubulin is assembled and thus only accessible at the tips of the MTs (Fig. 8, model C). However, our deletion analysis showed that both N-terminal and C-terminal deleted TPPP/p25 fragments retain their ability to bundle MTs. An alternative model is that TPPP/p225 may interact with 3 different sites (Fig. 8, model B). Our current domain analysis though, does not provide evidence for the presence of a third binding site. Therefore, we propose a dimer, like that proposed for the PRC1/MAP65-1 or the tau MT induced bundling57. These models are based on the ability of the MAP to form dimers that could crosslink two MTs. The issue of TPPP/p25 dimer formation has been proposed previously, and depending on GTP concentration and crosslinking conditions, TPPP/p25 dimers have been detected35. Our biophysical analysis of all the fragments though did not support the formation of TPPP/p25 dimers in solution. However, one could propose a conditional protein-protein interaction that would depend on MT binding (Fig. 8, models D, E and F). Full-length and the N-and C-terminal deleted fragments in our hands had very similar Kd for MT binding. However, the C-terminal deleted fragment exhibited a Bmax that was 50% lower than the full length and N-terminal deleted fragment. One hypothesis that can explain this difference is that the N- and C-termini bind to different sites and that the tubulin binding site for the C-terminal domain is masked in taxol stabilized MTs. One possible binding site for the C-terminus of TPPP/p25 may be at the tips of the MTs (Fig. 8, model F). Further analysis of the possible MT tip binding properties of the TPPP/p25 is needed.


Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling.

DeBonis S, Neumann E, Skoufias DA - Sci Rep (2015)

Schematic representation of the possible interaction modes of TPPP/p25 resulting in microtubule binding and bundling.The various elements and distances are not drawn to scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542545&req=5

f8: Schematic representation of the possible interaction modes of TPPP/p25 resulting in microtubule binding and bundling.The various elements and distances are not drawn to scale.
Mentions: The presence of two MT binding sites on TPPP/p25 provides an obvious model for how one molecule of TPPP/p25 can crosslink two MTs, with each binding site interacting with a different MT (Fig. 8, models A and C). Alternatively, one of the sites on TPPP/p25 might bind a tubulin site which is masked when tubulin is assembled and thus only accessible at the tips of the MTs (Fig. 8, model C). However, our deletion analysis showed that both N-terminal and C-terminal deleted TPPP/p25 fragments retain their ability to bundle MTs. An alternative model is that TPPP/p225 may interact with 3 different sites (Fig. 8, model B). Our current domain analysis though, does not provide evidence for the presence of a third binding site. Therefore, we propose a dimer, like that proposed for the PRC1/MAP65-1 or the tau MT induced bundling57. These models are based on the ability of the MAP to form dimers that could crosslink two MTs. The issue of TPPP/p25 dimer formation has been proposed previously, and depending on GTP concentration and crosslinking conditions, TPPP/p25 dimers have been detected35. Our biophysical analysis of all the fragments though did not support the formation of TPPP/p25 dimers in solution. However, one could propose a conditional protein-protein interaction that would depend on MT binding (Fig. 8, models D, E and F). Full-length and the N-and C-terminal deleted fragments in our hands had very similar Kd for MT binding. However, the C-terminal deleted fragment exhibited a Bmax that was 50% lower than the full length and N-terminal deleted fragment. One hypothesis that can explain this difference is that the N- and C-termini bind to different sites and that the tubulin binding site for the C-terminal domain is masked in taxol stabilized MTs. One possible binding site for the C-terminus of TPPP/p25 may be at the tips of the MTs (Fig. 8, model F). Further analysis of the possible MT tip binding properties of the TPPP/p25 is needed.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Université de Grenoble Alpes, F-38044 Grenoble, France.

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.

No MeSH data available.


Related in: MedlinePlus