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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

TPPP/p25 induces microtubule polymerization.(a) Schematic representation of the various TPPP/p25 fragments used in this study. The ΔN(49) fragments has the first 49 residues deleted whereas the ΔC(158) fragment has residues 158–219 deleted; core fragment lacks both residues 1–49 and 158–219. (b) Coomasie-stained gel of bacterially expressed and purified His-tagged TPPP/p25 fragments [FL = full length TPPP/p25; ΔN = ΔN(49); ΔC = ΔC(158)]. (c) Light scattering assays (OD350 nm) of tubulin solutions (15 μM) in the presence of equimolar concentrations of each of the four TPPP/p25 constructs. (d) EM images of microtubules assembled from tubulin solutions (15 μM) in the absence or presence of 15 μM or 30 μM of each of the four different TPPP/p25 fragments (scale bars = 50 nm).
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f1: TPPP/p25 induces microtubule polymerization.(a) Schematic representation of the various TPPP/p25 fragments used in this study. The ΔN(49) fragments has the first 49 residues deleted whereas the ΔC(158) fragment has residues 158–219 deleted; core fragment lacks both residues 1–49 and 158–219. (b) Coomasie-stained gel of bacterially expressed and purified His-tagged TPPP/p25 fragments [FL = full length TPPP/p25; ΔN = ΔN(49); ΔC = ΔC(158)]. (c) Light scattering assays (OD350 nm) of tubulin solutions (15 μM) in the presence of equimolar concentrations of each of the four TPPP/p25 constructs. (d) EM images of microtubules assembled from tubulin solutions (15 μM) in the absence or presence of 15 μM or 30 μM of each of the four different TPPP/p25 fragments (scale bars = 50 nm).

Mentions: One characteristic property of the TPPP/p25 protein following circular dichroism was shown to be its low alpha helical content1222. Consistent with this observation, bioinformatic analysis of the TPPP/p25 amino acid sequence using algorithms to detect folded and disordered protein domains predicted that the protein has disordered N-terminal (first 50 residues) and C-terminal (last 61 residues) regions (Fig. S1)2933. Furthermore, the NMR structure of TPPP/p20 (PDB entry 2JRF), another member of the TPPP protein family, revealed unstructured N- and C-termini and a folded central domain composed of five alpha helices25. TPPP/p20 has a shorter N-terminus and shares amino acid similarity in the central and C-terminal domains compared to TPPP/p2534. We also observed that bacterially expressed and purified full-length TPPP/p25 is proteolytically cleaved into different fragments after prolonged incubation with purified tubulin. N-terminal sequencing and MALDI protein mass spectrometry of the resulting peptides identified protein species truncated at either the N-terminus, C-terminus or both (Fig. S2). In view of these results and of the structural and bioinformatic data, we decided to make three TPPP/p25 deletion constructs, which lack either residues 1–49, C-terminal residues 158–219, or both these regions, denoted ΔN(49), ΔC(158), and “core”, respectively (Fig. 1a). The full-length (FL) and three truncated constructs of TPPP/p25 bear a C-terminal His tag for affinity purification following bacterial overexpression (Fig. 1b). TPPP/p25 has been previously reported to form dimers in solution35. However, analysis of all four purified constructs by analytical ultracentrifugation and HPLC-MALLS gave no evidence for the existence of dimers, suggesting that the constructs in our solution conditions are monomeric (data not shown).


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

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

TPPP/p25 induces microtubule polymerization.(a) Schematic representation of the various TPPP/p25 fragments used in this study. The ΔN(49) fragments has the first 49 residues deleted whereas the ΔC(158) fragment has residues 158–219 deleted; core fragment lacks both residues 1–49 and 158–219. (b) Coomasie-stained gel of bacterially expressed and purified His-tagged TPPP/p25 fragments [FL = full length TPPP/p25; ΔN = ΔN(49); ΔC = ΔC(158)]. (c) Light scattering assays (OD350 nm) of tubulin solutions (15 μM) in the presence of equimolar concentrations of each of the four TPPP/p25 constructs. (d) EM images of microtubules assembled from tubulin solutions (15 μM) in the absence or presence of 15 μM or 30 μM of each of the four different TPPP/p25 fragments (scale bars = 50 nm).
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f1: TPPP/p25 induces microtubule polymerization.(a) Schematic representation of the various TPPP/p25 fragments used in this study. The ΔN(49) fragments has the first 49 residues deleted whereas the ΔC(158) fragment has residues 158–219 deleted; core fragment lacks both residues 1–49 and 158–219. (b) Coomasie-stained gel of bacterially expressed and purified His-tagged TPPP/p25 fragments [FL = full length TPPP/p25; ΔN = ΔN(49); ΔC = ΔC(158)]. (c) Light scattering assays (OD350 nm) of tubulin solutions (15 μM) in the presence of equimolar concentrations of each of the four TPPP/p25 constructs. (d) EM images of microtubules assembled from tubulin solutions (15 μM) in the absence or presence of 15 μM or 30 μM of each of the four different TPPP/p25 fragments (scale bars = 50 nm).
Mentions: One characteristic property of the TPPP/p25 protein following circular dichroism was shown to be its low alpha helical content1222. Consistent with this observation, bioinformatic analysis of the TPPP/p25 amino acid sequence using algorithms to detect folded and disordered protein domains predicted that the protein has disordered N-terminal (first 50 residues) and C-terminal (last 61 residues) regions (Fig. S1)2933. Furthermore, the NMR structure of TPPP/p20 (PDB entry 2JRF), another member of the TPPP protein family, revealed unstructured N- and C-termini and a folded central domain composed of five alpha helices25. TPPP/p20 has a shorter N-terminus and shares amino acid similarity in the central and C-terminal domains compared to TPPP/p2534. We also observed that bacterially expressed and purified full-length TPPP/p25 is proteolytically cleaved into different fragments after prolonged incubation with purified tubulin. N-terminal sequencing and MALDI protein mass spectrometry of the resulting peptides identified protein species truncated at either the N-terminus, C-terminus or both (Fig. S2). In view of these results and of the structural and bioinformatic data, we decided to make three TPPP/p25 deletion constructs, which lack either residues 1–49, C-terminal residues 158–219, or both these regions, denoted ΔN(49), ΔC(158), and “core”, respectively (Fig. 1a). The full-length (FL) and three truncated constructs of TPPP/p25 bear a C-terminal His tag for affinity purification following bacterial overexpression (Fig. 1b). TPPP/p25 has been previously reported to form dimers in solution35. However, analysis of all four purified constructs by analytical ultracentrifugation and HPLC-MALLS gave no evidence for the existence of dimers, suggesting that the constructs in our solution conditions are monomeric (data not shown).

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