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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

Quantitative analysis of the TPPP/p25 binding interaction to taxol stabilized microtubules.Binding curves with 1.5 μM GFP-tagged TPPP/p25 (WT or ΔN, ΔC) at increasing MT concentrations. Each data point represents the mean ± SD from three independent experiments.
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f4: Quantitative analysis of the TPPP/p25 binding interaction to taxol stabilized microtubules.Binding curves with 1.5 μM GFP-tagged TPPP/p25 (WT or ΔN, ΔC) at increasing MT concentrations. Each data point represents the mean ± SD from three independent experiments.

Mentions: We then proceeded to quantify the fluorescence signal of the bound GFP-TPPP/p25 fragments from the MT pellets and the unbound in the supernatant fraction after incubation of 1.5 μM TPPP/p25 with increasing concentrations of taxol stabilized MTs (Fig. 4). Following this experimental approach the apparent Kd for the three TPPP/p25 fragments was determined to be 1.44 ± 0.26 μM for the full length protein, 0.86 ± 0.15 μM for ΔN(49) and 1.75 ± 1.03 μM for the ΔC(158) (Table 1). Given the observation that MTs bundle in the presence of TPPP/p25 the apparent Kd is likely to be a composite of MT binding and TPPP/p25 dimerization or oligomerization. A notable difference was observed in the Bmax for the C-terminal deleted fragment ΔC(158), which was 50% of that of the full length or ΔN(49) fragment (Table 1).


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

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

Quantitative analysis of the TPPP/p25 binding interaction to taxol stabilized microtubules.Binding curves with 1.5 μM GFP-tagged TPPP/p25 (WT or ΔN, ΔC) at increasing MT concentrations. Each data point represents the mean ± SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Quantitative analysis of the TPPP/p25 binding interaction to taxol stabilized microtubules.Binding curves with 1.5 μM GFP-tagged TPPP/p25 (WT or ΔN, ΔC) at increasing MT concentrations. Each data point represents the mean ± SD from three independent experiments.
Mentions: We then proceeded to quantify the fluorescence signal of the bound GFP-TPPP/p25 fragments from the MT pellets and the unbound in the supernatant fraction after incubation of 1.5 μM TPPP/p25 with increasing concentrations of taxol stabilized MTs (Fig. 4). Following this experimental approach the apparent Kd for the three TPPP/p25 fragments was determined to be 1.44 ± 0.26 μM for the full length protein, 0.86 ± 0.15 μM for ΔN(49) and 1.75 ± 1.03 μM for the ΔC(158) (Table 1). Given the observation that MTs bundle in the presence of TPPP/p25 the apparent Kd is likely to be a composite of MT binding and TPPP/p25 dimerization or oligomerization. A notable difference was observed in the Bmax for the C-terminal deleted fragment ΔC(158), which was 50% of that of the full length or ΔN(49) fragment (Table 1).

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