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Tubulin binding and polymerization promoting properties of TPPP proteins are evolutionarily conserved Judit Oláh, Tibor Szénási, Adél Szabó, Kinga Kovács, Péter L#w, Mauro Stifanic, and Ferenc Orosz Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00902 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Tubulin binding and polymerization promoting properties of TPPP proteins are evolutionarily conserved Judit Oláh†, Tibor Szénási†, Adél Szabó†, Kinga Kovács†, Péter Lőw‡, Mauro Štifanić§, Ferenc Orosz*† †

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences; Magyar ‡

tudósok körútja 2., Budapest, H-1117, Hungary. Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University; Pázmány Péter sétány 1/C., Budapest, H-1117, Hungary. §Department of Natural and Health Studies, Juraj Dobrila University of Pula; Zagrebačka 30, HR-52100 Pula, Croatia.

Corresponding author: Ferenc Orosz Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences Magyar tudósok körútja 2. H-1117 Budapest, Hungary Phone: (36)-1-3826714 E-mail address: [email protected]

Funding information: M.Š thanks to Ministry of Science, Education and Sports of the Republic of Croatia for institutional funding of scientific activities in public universities and public research institutes grant to the University Juraj Dobrila of Pula. T. Sz. was supported by a Junior Scientist Grant of the Hungarian Academy of Sciences, FK-078/2014. The research was supported by Hungarian National Scientific Research Fund Grant OTKA, K-112144 (J. O., F. O).

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Abbreviations: TPPP - Tubulin Polymerization Promoting Protein; IUP - intrinsically unstructured protein; MAP - microtubule associated protein; TFE - 2,2,2-trifluoroethanol.


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Abstract Tubulin Polymerization Promoting Proteins (TPPPs) constitute a eukaryotic protein family. There are three TPPP paralogs in the human genome; denoted as TPPP1, TPPP2 and TPPP3. TPPP1 and TPPP3 are intrinsically unstructured proteins (IUPs) that bind and polymerize tubulin and stabilize microtubules but TPPP2 does not. Vertebrate TPPPs originated from the ancient invertebrate TPPP by two-round wholegenome-duplication thus it was an open question whether the tubulin/microtubule binding function of TPPP1 and TPPP3 is a newly acquired property or it was present in the invertebrate orthologs (generally one TPPP/species). To answer this question, we investigated a TPPP from a simple and early branching animal, the sponge Suberites domuncula. Bioinformatics, biochemical, immunochemical, spectroscopic and electron microscopic data showed that the properties of sponge protein correspond to those of TPPP1, namely, it is an IUP which binds strongly tubulin and induces its polymerization proving that these features of animal TPPPs are evolutionary conserved.


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Microtubules are cytoskeletal filaments composed of α and β tubulin that play a role in cell migration, establish cell polarity, serve as intracellular transport tracks, and form the mitotic spindle. Microtubules are formed by the polymerization of αβ-tubulin heterodimers into tube-like polymers, the functions of which are mediated by various factors including their interacting partner proteins (1). These microtubule associated proteins (MAPs) regulate the rates of polymerization and depolymerization in space and time, often in a cell type specific manner (2, 3). For example, two of the most studied MAPs, tau and MAP2, are expressed predominantly in neurons; in the thin, long axon there are tau-bound microtubules, whereas in the multiple, shorter dendrites MAP2-bound microtubules can be found. A few MAPs bind not only to microtubules but also to tubulin dimers. For example, XMAP215 family proteins (4) bind both to tubulin dimers and microtubules via their TOG domains (5) and promote the polymerization of the dimers into microtubules (6). Some members of a eukaryotic protein family, the Tubulin Polymerization Promoting Proteins (TPPPs), have similar properties (7, 8). Its first member was originally found as a brain specific protein with a mass of 25 kDa during the isolation of a protein kinase (9); independently it was isolated from bovine brain and denoted as Tubulin Polymerization Promoting Protein, TPPP/p25 based on its molecular mass and in vitro function (10). Moreover, it also bundles microtubules and increases their acetylation level thus affects their dynamics and ultrastructure as a MAP (10-14). The tubulin binding site of human TPPP1 was predicted to be localized on its C-terminus based on the comparison of its sequence with the microtubule binding domain of tau (12), which was verified later experimentally (15). TPPP/p25 is predominantly expressed in oligodendrocytes in the brain; it is indispensable for their differentiation (1619). It co-localizes with α-synuclein in inclusion bodies in Parkinson’s disease and multiple system atrophy (20, 21). An outstanding characteristics of this basic protein is that it belongs to the intrinsically unstructured/disordered proteins (IUPs/IDPs) (20, 21), similarly to other microtubule binding proteins, tau and MAP2c (22-24). TPPP/p25 might work as a protective factor of cells against the damage effects of abnormal prion protein accumulation (25). The three human TPPP paralogs are denoted as TPPP/p25, TPPP2/p18 and TPPP3/p20 (shortly TPPP1, TPPP2 and TPPP3, respectively), according to their molecular masses (7). The pairwise sequence


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comparisons of the three proteins show 63.6, 61.2 and 57.7% identities for TPPP1-TPPP3, TPPP3-TPPP2, and TPPP1-TPPP2, respectively (7). The properties of TPPP2 differ significantly from those of the other human paralogs. Human TPPP1 and TPPP3 bind and stabilize microtubules displaying MAP-like features but TPPP2 does not. TPPP1 is a brain-specific protein which is supported by its function as well; TPPP3 was also isolated from brain (7) but TPPP2 does not occur in adult mammalian brain (26). A further difference is that TPPP1 and TPPP3 are IUPs, whereas, based on indirect proofs, TPPP2 is not (7). Thus one can hypothesize an (un)structure – function relationship of TPPP proteins. The 51-219 amino acids in TPPP1 are denoted as p25alpha domain, Pfam05517 or IPR008907, which is common in the three proteins and corresponds practically to the whole sequence of TPPP2 or TPPP3. We suggested that the single invertebrate tppp gene diversified into tppp1 and the precursor of tppp2/tppp3 in the first round of the tworound whole-genome duplication occurred in the early vertebrate lineage (27). We introduced the term ‘Tubulin Polymerization Promoting Protein (TPPP)-like proteins’ (28). Proteins involving the p25alpha domain belong to this eukaryotic protein superfamily named after its first member, TPPP/p25. TPPP-like proteins can be further grouped according to either the length of their p25alpha domain (long, short, truncated or partial), or the occurrence of additional domain(s). The phylogenetic distribution is characteristic for the different subfamilies: e.g., Opisthokonta (Metazoa, Fungi as well as Choanomonada and some other unicellular organisms) almost exclusively encode the long-type TPPPs. Long-type TPPPs are characterized by the presence of a Rossmann-like sequence (GxGxGxxGR) in their C-terminal part. Functional data are available only for the two human paralogs, TPPP1 and TPPP3, which play a role in developmental processes of the brain (15, 19) and the musculoskeletal system (29), respectively. The vertebrate TPPPs originated from the ancient invertebrate TPPP by two-round whole genome duplication, but an open question remains whether the microtubule binding/stabilizing function of TPPP1/TPPP3 is a newly acquired property or it was already present in the invertebrate orthologs (generally one TPPP/species). In general: Are the characteristics of the long-type TPPPs present in invertebrate animals more similar to TPPP1/TPPP3 or to TPPP2? Thus our aim was to investigate and characterize a TPPP from a simple and early branching animal, the sponge Suberites domuncula.


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Oligonucleotides and plasmid construction The Suberites TPPP with a C-terminal His-tag was amplified by PCR using forward primer 5'CTCATATGGCTACAGGATTGGAATCTACC-3'

and

reverse

primer

5'-

GCCTCGAGCTTCTTCTTGTCGTAAGTTCCC-3' and pUC57-6HisTPPP (Protogenix) as a template. The C-terminal truncated Suberites TPPP by residues 129–180 (Suberites TPPP ∆129–180) was amplified by PCR using forward primer 5'-CTCATATGGCTACAGGATTGGAATCTACC-3' and reverse primer 5'CCTTCTCGAGACCGGTGTACTTAGAAGTGTC-3' and pUC57-6HisTPPP as a template. Inserts were digested with NdeI and XhoI restriction enzymes, then ligated into pET21c (Novagen) prokaryotic expression vector. The construct was verified by restriction enzyme analysis and DNA sequencing. Human recombinant TPPP/p25 was cloned as described previously (14). Protein purification Suberites and human recombinant TPPPs with a His-tag was expressed in E. coli BL21 (DE3) cells and isolated on HIS-Select™ Cartridge (Sigma-Aldrich) similarly as in (7). The following extinction coefficients were used to determine protein concentration from the absorbance at 280 nm: 7450 M−1 *cm−1, 2980 M−1 *cm−1 and 10095 M−1 *cm−1 for sponge, truncated sponge and human TPPPs, respectively. Tubulin was prepared from bovine brain (30). CD spectroscopy CD measurements were carried out as described previously (15) using a Jasco J-720 spectropolarimeter. The concentration of proteins was 4.5 µM for TPPP forms and 0.9 µM for tubulin. Mean molar ellipticity (MRE) per residue was calculated from the measured ellipticity (Θm) as described in (15). The content of αhelix was determined from the MRE value at 222 nm according to (31): % α-helix = (|MRE222| − 2340) ⁄ 303.

The

secondary

structures

were

also

estimated

using

the

DichroWeb

server

(http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) (32) using the CONTINLL algorithm (33, 34) and the SET7 reference set.


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ELISA ELISA experiments were carried out similarly as described previously (15). Here the plate was coated with 5 µg/ml TPPP forms, and then it was incubated with serial dilutions of tubulin for 1 h. Then the plate was incubated with tubulin antibody (1:5000, Sigma-Aldrich T9026 monoclonal α-tubulin antibody produced in mouse, clone DM1A, lot# 034M4792V), followed by a secondary IgG conjugated with peroxidase (1:5000, Sigma-Aldrich). Absorbance was read at 490 nm with an EnSpire multimode reader (Perkin Elmer). No signal was detected if tubulin or any of the antibodies were omitted. Pelleting The measurements were carried out as described in (15). The concentration of tubulin was 5 µM, while that of the various TPPP forms was 10 µM. Polymerization The assembly of tubulin (6 µM) was followed in polymerization buffer (50 mM 2-(Nmorpholino)ethanesulfonic acid, 100 mM KCl, 1 mM dithioerythritol, 1 mM MgCl2 and 1 mM ethylene glycol tetraacetic acid, pH 6.6) at 37 °C. The tubulin polymerization was induced by the addition of the various human and sponge TPPP forms, respectively. The attenuance was monitored at 350 nm by a Cary 100 spectrophotometer (Varian). Sample preparation for electron microscopy Samples were prepared at two different conditions. (a) Tubulin (6 µM) polymerization was induced by 3 µM and 6 µM of the human and the sponge TPPP forms, respectively, without paclitaxel and centrifuged at 25,000 g for 20 min at 37°C. (b) Tubulin at 12 µM was preassembled in polymerization buffer containing 20 µM paclitaxel at 37°C and the increase of the attenuance was monitored at 350 nm until it reached a constant value. Then human or sponge TPPPs were added, in a final concentration of 3 µM and 6 µM, respectively. The increase of the attenuance was monitored at 350 nm until it reached a constant value and the samples were centrifuged as above.


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Transmission electron microscopy For negative staining, a drop of a sample from the polymerization assay without centrifugation was applied to formvar-coated copper grids for 60 s. After the removal of the solution, the grid was stained with a drop of freshly filtered 1% aqueous uranyl acetate for 40 s. Blotting with filter paper was used to remove the excess stain. For routine electron microscopy, microtubule-containing samples were pelleted by centrifugation, and the pellets were prefixed in a mixture of 2% glutaraldehyde, 0.2% tannic acid in 0.1 M sodium cacodylate, pH 7.4, for 1 h at room temperature and for 24 h at 4°C, then postfixed in 0.5% OsO4 for 1 h and embedded in Durcupan (Fluka, Switzerland). Thin sections were contrasted with lead citrate and examined and photographed in a JEOL JEM-1011 electron microscope, operating at 60kV, equipped with Olympus Morada CCD camera using Olympus iTEM (TEM imaging platform) software. Structural prediction Prediction of unstructured regions: Sequences were submitted to the VSL2B server optimized for proteins containing

both

structure

and

disorder

(35)

and

freely

available

at

http://www.dabi.temple.edu/disprot/predictorVSL2.php. The IUPRED server (36), freely available at http://iupred.enzim.hu/, was also used, in the “long disorder” mode. Prediction of binding motifs: disordered

binding

regions

in

S.

domuncula

were

predicted

by

the

Anchor

program

(http://anchor.enzim.hu/) (37). The Clustal Omega program (38) was used for multiple alignments of sequences.

Results and Discussion The vast majority of animals belong to the Eumetazoa. To our knowledge, all eumetazoans, whose genomes were fully sequenced, contain at least one (long-type) TPPP protein. The less developed phylum Placozoa does not have it. Instead, the representative member of this group, Trichoplax adhaerens, possesses a related protein, apicortin, which contains a partial p25alpha domain as a part of a multidomain protein (39). However, TPPP sequences were found in the third big groups of animals, sponges, in Amphimedon queenslandica (XP_003384590) and in S. domuncula (ADX30619) (40). Sponges (Porifera)


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are one of the oldest metazoan phyla, which are highly conservative and often considered as the living fossils of multicellular animals. They are of special importance and often used as model organism for evolutionary, developmental and functional studies of Metazoa (41). Similarly to TPPP2 and TPPP3, sponge TPPPs do not have the N-terminal part of human TPPP1 but their C-terminal regions do share high sequence similarity with the human one (Fig. 1). The pairwise sequence identities of the full-length human and sponge TPPPs vary between 42% and 53% which is only slightly less than the identity of the three human paralogs. The identity is the highest among the C-termini, except the 11 extra amino acids being present inside the C-terminus of sponge proteins, which are missing in TPPPs of other animals, including the three human paralogs. It is unlikely that this part is an insertion in sponge TPPPs; it is more likely that it was lost in all other animals since it is present in the TPPP (XP_001743131) of a Choanomonada, Monosiga brevicollis, a unicellular Opisthokonta.

The IUP nature of sponge TPPPs Our previous experiments using human TPPP1 suggested that the intrinsically disordered nature of human TPPP1 is a prerequisite of its strong tubulin binding property. Thus we used disorder prediction methods to determine whether sponge TPPPs may belong to IUPs (Fig. 2). (We use the term IUP as defined by The Database of Protein Disorder (DisProt): a protein that contains at least one disordered region (42).) Both used methods suggest that the C-terminal part of these proteins is intrinsically disordered, similarly to human TPPP1, where the disordered N- and C-termini straddle a middle flexible segment (43); and to TPPP3, which shows a highly ordered α-helical structure of amino acids 8-102, and a disordered 103-178 region (44), as showed in both cases by NMR. It is worth mentioning that neither the sponge TPPPs nor the human TPPP3 contain the long disordered N-terminus of human TPPP1. A common feature of many IUPs is that their structures can be ordered due to their binding to targets. Experimental data show that the majority of IUPs fold upon binding to their physiological partners (45). The inherent secondary structure preferences of IUPs are characterized by a tendency to form α-helixes rather than β-sheets (46). The solvent 2,2,2-trifluoroethanol (TFE) mimics the hydrophobic environment experienced by proteins in protein–protein interactions and is therefore widely used as a probe to discover 9 ACS Paragon Plus Environment

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regions that have a propensity to undergo an induced folding into helices (47, 48). It has recently been shown that TFE induces disorder-to-helix transitions in many IUPs possessing “Anchor regions” (48) (see the next paragraph). We have studied the effect of TFE on the structure of S. domuncula TPPP. Its CD spectrum showed an increased α-helicity upon the addition of TFE, as indicated by the characteristic minimum at 222 nm and by the estimated α-helix content using different methods (Fig. 3 and Table 1). Thus the use of TFE reveals an α-helix forming potential of this IUP, similarly as observed with human TPPP1 (18).

Binding of S. domuncula TPPP to tubulin To test the association of the sponge TPPP with tubulin, CD measurements were performed. The addition of tubulin to the sponge ortholog resulted in a decreased, but still significant positive difference in the ellipticity spectra, as compared to the human TPPP1 (5) (Fig. 4A,B). This change in conformation indicates an interaction between the tubulin and these TPPP orthologs, however, as the change in secondary structure content of each protein within the mixtures cannot be determined, because we do not know whether only TPPP or both proteins undergo a conformational change during the interaction, the nature of this conformation change cannot be precisely described. The binding affinities of the human and Suberites TPPPs to tubulin were determined in an ELISA assay. The immobilized TPPP forms were titrated with tubulin and complex formation was detected using a specific tubulin antibody. The results revealed that tubulin bound to both the human and the Suberites TPPP forms (Fig. 4C). The binding affinities of human TPPP1 and Suberites TPPP to tubulin were evaluated by curve fitting, simple hyperbolic saturation was assumed, and the values were found to be 38 ± 6 and 30 ± 0.6 nM, respectively. The data fit to the value, 10.5 nM, determined earlier by ELISA for human TPPP1 (49). The similar binding strength can be explained by the fact that the tubulin binding motif of human TPPP1 was localized within the disordered C-terminal part (15) and that the Anchor program predicted a binding motif (“Anchor region”) in the rather similar and also disordered C-terminus of S. domuncula (cf. Fig. 2). The necessity of the disordered region for tubulin binding and polymerization was shown earlier in the case of the human TPPP1 (15). To address this question in the case of the sponge TPPP, a C-terminal 10 ACS Paragon Plus Environment

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truncated sponge TPPP (∆129-180) was constructed; the deletion started at a point analogous to that used in studies of the human TPPP1 (15). Pelleting experiments showed that S. domuncula TPPP binds to tubulin in a similar manner as human TPPP1 (Fig. 5A). By themselves, tubulin and either human or sponge TPPPs can be found entirely in the supernatant fraction, with no protein observed in the pellet. However, when tubulin is pre-incubated with either human or sponge TPPP, the tubulin and TPPP proteins are distributed between both the pellet and supernatant fractions, indicating their interaction and the polymerization of tubulin due to the effect of TPPP proteins. However, the truncated sponge TPPP practically does not induce tubulin polymerization (Fig. 5A). The characteristic tubulin polymerization promoting activity of TPPPs, as monitored by recording turbidimetry, was observed upon addition of full-length TPPP, but not upon addition of truncated S. domuncula TPPP (Fig. 5B). Compared to the human TPPP1, double concentration of the sponge protein was needed to achieve the same polymerizing effect. The C-terminal truncated sponge TPPP did not induce tubulin polymerization even in quadruple concentration. Earlier we found that the C-terminal truncated human TPPP1 displayed much less polymerization promoting activity than the native one (15). The real control of the experiment presented here was the TPPP1 truncated in both its N- and C-termini, which had no polymerizing effect at all (15), since wild type sponge TPPP does not possess the long unstructured Nterminal part of the human TPPP1. If the full-length human and sponge TPPPs were added to preassembled paclitaxel-stabilized microtubules, the turbidity of the samples significantly increased further (Fig. 5C). These findings indicate that S. domuncula TPPP can induce, on one hand, tubulin polymerization and, on the other hand, alterations in the ultrastructure of microtubule assemblies.

Electron microscopy of TPPP-induced tubular assemblies To visualize the morphological consequences of the interaction between sponge TPPP and tubulin/microtubules observed in turbidimetry and pelleting assays (cf. Figure 5), electron microscopic studies were carried out on negatively stained preparations and on sections from resin-embedded pellets. We showed earlier that mammalian (human and bovine) TPPP1 induces formation of both protein aggregates and microtubules from tubulin, and the microtubules formed are often arranged into bundles (7, 11 ACS Paragon Plus Environment

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10, 11). S. domuncula TPPP produces very similar forms, single and bundled microtubules and amorphous aggregates (Fig. 6). TPPPs, human or sponge, were added to tubulin, and the pellet fraction was sedimented at 25,000g and studied in the electron microscope. The population of microtubules assembled in the presence of TPPPs consists of a straight-running single microtubule or bundled microtubules (Fig. 6), most of which are often curved and shorter than those observed in the case of paclitaxel-stabilized microtubules (Fig. 7).

Binding of S. domuncula TPPP to microtubules and its effect on microtubule organization In another set of experiments, we elucidated whether sponge TPPP is able to bundle paclitaxel stabilized microtubules forming larger particles. Paclitaxel-stabilized microtubules were prepared, then incubated with TPPP, human or sponge, and sedimented at 25,000g. The control sample did not contain TPPP. The electron microscopic images of the pelleted microtubules are shown in Fig. 7. The control sample contains loosely arranged single microtubules. The TPPP-containing samples are crowded with bundles of microtubules consisting of many wall-to-wall contacted tubules (Fig. 7). This bundling can explain the observed increase in turbidity when TPPPs were added to the paclitaxel-stabilized microtubules (see Figure 5). The bundling is probably very fast as seen from the rapid elevation of the turbidity level. The presented results clearly show that, despite the big phylogenetic distance, sponge TPPP possesses the same tubulin binding and polymerizing properties as human TPPP1. This finding means that tubulin binding and polymerization promoting features of long-type TPPPs are evolutionary ancient and well conserved in animals. They are original characteristics of this protein which were preserved in mammalian TPPP1 and TPPP3 but lost in TPPP2. This kind of sub-functionalization is a frequent phenomenon among paralogs arisen during the two-round whole-genome duplication in the early vertebrate lineage, which helped the redundant paralogs to “survive” (50).

Acknowledgements We thank Sarolta Pálfia for skillful technical assistance. 12 ACS Paragon Plus Environment

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Declaration of interest: The authors declare that they have no conflicts of interest with the contents of this article.


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11. Tirián, L., Hlavanda, E., Oláh, J., Horváth, I., Orosz, F. Szabó, B., Kovács, J., Szabad J., and Ovádi, J. (2003) TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation, Proc. Natl. Acad. Sci. USA 100, 13976–13981. 12. Hlavanda, E., Klement, E., Kókai, E., Kovács, J., Vincze, O., Orosz, F., Medzihradszky, K. F., Dombrádi, V., and Ovádi, J. (2007) Phosphorylation blocks the activity of tubulin polymerizationpromoting protein (TPPP): identification of sites targeted by different kinases, J. Biol. Chem. 282, 29531– 29539. 13. Lehotzky, A., Tirián, L., Tőkési, N., Lénárt, P., Szabó, B., Kovács, J., and Ovádi, J. (2004) Dynamic targeting of microtubules by TPPP/p25 affects cell survival, J. Cell Sci. 117, 6249–6259. 14. Tőkési, N., Lehotzky, A., Horváth, I., Szabó, B., Oláh, J., Lau, P., and Ovádi, J. (2010) TPPP/p25 promotes tubulin acetylation by inhibiting histone deacetylase 6, J. Biol. Chem. 285, 17896–17906. 15. Tőkési, N., Oláh, J., Hlavanda, E., Szunyogh, S., Szabó, A., Babos, F., Magyar, A., Lehotzky A., Vass, E., and Ovádi, J. (2014) Identification of motives mediating alternative functions of the neomorphic moonlighting TPPP/p25, Biochim. Biophys. Acta 1842, 547–557. 16. Takahashi, M., Tomizawa, K., Fujita, S. C., Sato, K., Uchida, T., and Imahori, K. (1993) A brainspecific protein p25 is localized and associated with oligodendrocytes, neuropil, and fiber-like structures of the CA hippocampal region in the rat brain, J. Neurochem. 60, 228–235. 17. Skjoerringe, T., Lundvig, D. M. S., Jensen, P. H., and Moos, T. (2006) P25 alpha/tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain, J. Neurochem. 99, 333–342. 18. Lehotzky, A., Tőkési, N., Gonzalez-Alvarez, I., Merino, V., Bermejo, M., Orosz, F., Lau P., Kovacs, G. G., and Ovádi, J. (2008) Progress in the development of early diagnosis and a drug with unique pharmacology to improve cancer therapy, Philos. Transact. A Math. Phys. Eng. Sci. 366, 3599–3617. 19. Lehotzky, A., Lau, P., Tőkési, N., Muja, N., Hudson, LD., and Ovádi, J. (2010) Tubulin polymerization-promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation, Glia 58, 157– 168. 15 ACS Paragon Plus Environment

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20. Kovács, G. G., László, L., Kovács, J., Jensen, P. H., Lindersson, E., Botond, G., Molnár, T., Perczel, A., Hudecz, F., Mezo, G., Erdei, A., Tirián, L., Lehotzky, A., Gelpi, E., Budka, H., and Ovádi, J. (2004) Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alphasynucleinopathies, Neurobiol. Dis. 17, 155–162. 21. Orosz, F., Kovács, G. G., Lehotzky, A., Oláh, J., Vincze, O., and Ovádi, J. (2004) TPPP/p25: from unfolded protein to misfolding disease: prediction and experiments, Biol. Cell 96, 701–711. 22. Tompa, P. (2002) Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527-533. 23. Csizmók, V., Bokor, M., Bánki, P., Klement, E., Medzihradszky, K.F., Friedrich, P., Tompa, K., Tompa, P. (2005) Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein 2. Biochemistry 44, 3955-3964. 24. Skrabana, R., Sevcik, J., Novak, M. (2006) Intrinsically disordered proteins in the neurodegenerative processes: formation of tau protein paired helical filaments and their analysis. Cell Mol. Neurobiol. 26, 1085-1097. 25. Zhou, R. M., Jing, Y. Y., Guo, Y., Gao, C., Zhang, B. Y., Chen, C., Shi, Q., Tian, C., Wang, Z. Y., Gong, H. S., Han, J., Xu, B. L., and Dong, X. P. (2011) Molecular interaction of TPPP with PrP antagonized the CytoPrP-induced disruption of microtubule structures and cytotoxicity, PLoS One 6, e23079 26. Zhang, Z., Wu, C., Huang, W., Wang, S., Zhao, E., Huang, Q., Xie, Y., and Mao, Y. (2002) A novel human gene whose product shares homology with bovine brain-specific protein p25 is expressed in fetal brain but not in adult brain, J. Hum. Genet. 47, 266-268. 27. Orosz, F. (2012) A fish-specific member of the TPPP protein family? J. Mol. Evol. 75, 55-72. 28. Orosz, F. (2012) A new protein superfamily: TPPP-like proteins, PLoS ONE 7, e49276 29. Staverosky, J. A., Pryce, B. A., Watson, S. S., and Schweitzer, R. (2009) Tubulin polymerizationpromoting protein family member 3, Tppp3, is a specific marker of the differentiating tendon sheath and synovial joints, Dev. Dyn. 238, 685-692.


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30. Na, C. N., and Timasheff, S. N. (1986) Interaction of vinblastine with calf brain tubulin multiple equilibria, Biochemistry 25, 6214-6222. 31. Chen,Y.-H., Yang, J.T., Martinez, H.M. (1972) Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry 11, 4120–4131. 32. Whitmore, L. and Wallace, B.A. (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89, 392-400. 33. Provencher, S.W. and Glockner, J. (1981) E stimation of globular protein secondary structure from circular dichroism. Biochemistry 20, 33-37. 34. Van Stokkum, I.H.M., Spoelder, H.J.W., Bloemendal, M., Van Grondelle, R., and Groen, F.C.A. (1990) Estimation of protein secondary structure and error analysis from CD spectra. Anal. Biochem. 191, 110118. 35. Obradovic, Z., Peng, K., Vucetic, S., Radivojac, P., and Dunker, A. K. (2005). Exploiting heterogeneous sequence properties improves prediction of protein disorder proteins, Proteins 61(S7), 176182. 36. Dosztányi, Z., Csizmók, V., Tompa, P., and Simon, I. (2005) IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content, Bioinformatics 21, 34333434. 37. Mészáros B., Simon, I., and Dosztányi, Z. (2009) Prediction of protein binding regions in disordered proteins, PLoS Comput. Biol. 5, e1000376 38. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J. D., and Higgins, D. G. (2011) Fast, scalable generation of highquality protein multiple sequence alignments using Clustal Omega, Mol. Syst. Biol. 7, 539. 39. Orosz, F. (2009) Apicortin, a unique protein, with a putative cytoskeletal role, shared only by apicomplexan parasites and the placozoan Trichoplax adhaerens, Infect. Genet. Evol. 9, 1275–1286.


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40. Stifanic, M., Batel, R., and Müller, W. E. G. (2011) Tubulin polymerization promoting protein (TPPP) ortholog from Suberites domuncula and comparative analysis of TPPP/p25 gene family, Biologia 66, 111– 120. 41. Müller, W. E. G. (1998) Origin of Metazoa: sponges as living fossils, Naturwissenschaften 85, 11-25. 42. Sickmeier, M., Hamilton, J. A., LeGall, T., Vacic, V., Cortese, M. S., Tantos, A., Szabo, B., Tompa, P., Chen, J., Uversky, V. N., Obradovic, Z., and Dunker, A. K. (2007) DisProt: the database of disordered proteins, Nucleic Acids Res. 35, D786–D793. 43. Zotter, A., Bodor, A., Oláh, J., Hlavanda, E., Orosz, F., Perczel, A., and Ovádi, J. (2011) Disordered TPPP/p25 binds GTP and displays Mg(2+)-dependent GTPase activity, FEBS Lett. 585, 803–808. 44. Aramini, J. M., Rossi, P., Shastry, R., Nwosu, C., Cunningham, K., Xiao, R., Liu, J., Baran, M. C., Rajan, P. K., Acton, T. B., Rost, B., Montelione, G. T. (2007) Solution NMR Structure of Tubulin Polymerization-promoting Protein Family Member 3 From Homo Sapiens. Northeast Structural Genomics target HR387, http://www.rcsb.org/pdb/explore/explore.do?pdbId=2JRF 45. Dyson, H. J., and Wright, P. E., (2002) Coupling of folding and binding for unstructured proteins, Curr. Opin. Struct. Biol. 12, 54–60. 46. Fuxreiter, M., Simon, I., Friedrich, P., and Tompa, P. (2004) Preformed structural elements feature in partner recognition by intrinsically unstructured proteins, J. Mol. Biol. 338, 1015–1026. 47. Hua, Q. X., Jia, W. H., Bullock, B. P., Habener, J. F., and Weiss, M. A. (1998) Transcriptional activator–coactivator recognition: nascent folding of a kinase-inducible transactivation domain predicts its structure on coactivator binding, Biochemistry 37, 5858–5866. 48. Glover, K., Mei, Y., Sinha S. C. (2016) Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions, Biochim. Biophys. Acta 1864, 1455-1463. 49. Oláh, J., Vincze, O., Virók, D., Simon, D., Bozsó, Z., Tőkési, N., Horváth, I., Hlavanda, E., Kovács, J., Magyar, A., Szűcs, M., Orosz, F., Penke, B., and Ovádi, J. (2011) Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein, J. Biol. Chem. 286, 34088–34100. 18 ACS Paragon Plus Environment

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50. Dehal, P., and Boore, J. L. (2005) Two rounds of whole genome duplication in the ancestral vertebrate, PLoS Biol 3, e314


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Tables Table 1. Estimated secondary structure content of sponge TPPP in the presence of TFE. TFE, %

α-helix, %

α-helix, %

based on Chen et al. (31)

β-strand, %

turn +disordered, %

according to DichroWeb using CONTINLL

0

15

23

23

55

20

25

33

14

53

40

27

40

14

47

60

41

54

5

40

For the estimation of the secondary structures, the measured CD spectra of sponge TPPP (Fig. 3) in the presence of various TFE concentrations were used. The α-helix content was calculated from either the MRE value at 222 nm based upon the method of Chen et al. (31) or was estimated using the DichroWeb server (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) (32) with the CONTINLL algorithm (33, 34) and the SET7 protein reference set.

Figure legends Figure 1. Multiple sequence alignment of TPPP-proteins by Clustal Omega (38). The alignment was refined manually. Hs1, Homo sapiens TPPP1 (NP_008961); Hs2, Homo sapiens TPPP2 (NP_776245); Hs3, Homo sapiens TPPP3 (NP_057224); Sd, Suberites domuncula TPPP (ADX30619); Aq, Amphimedon queenslandica TPPP (XP_003384590). Amino acid residues identical and similar in at least all but one protein are indicated by black and grey backgrounds, respectively. The asterisks indicate the Rossmann-like motif; the x labels the beginning of the p25alpha domain. Amino acids in the last line were truncated in the Suberites TPPP ∆129–180 (this study) or in the human TPPP1 ∆175–219 (Ref. 39) constructs, respectively. Figure 2. Disorder prediction of sponge TPPPs using the VSL2B predictor: Suberites domuncula ADX30619 (solid line) Amphimedon queenslandica XP_003384590 (dashed line). Disorder prediction values for the given residues are plotted against the amino acid residue number. The significance threshold, above which a residue is considered to be disordered, set to 0.5, is shown (dotted line). At the 20 ACS Paragon Plus Environment

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bottom of the figure, the binding site on S. domuncula protein predicted by ANCHOR is indicated (aa 156-171). Figure 3. Effect of TFE on the CD spectrum of Suberites domuncula TPPP (6 µM). The concentration of TFE was 0, 20, 40 and 60 % (solid, dashed, dotted, dash-dot-dotted line, respectively.) Figure 4. Binding of the human and sponge TPPP to tubulin. (A-B) CD spectroscopy in the case of human TPPP1 (A) and sponge TPPP (B). The ellipticities of tubulin (dash-dotted line) and the TPPP forms (bold line) were subtracted from the measured ellipticity of their mixtures (dashed line), which corresponds to the difference ellipticity shown (solid line). The concentration of tubulin and the TPPP forms was 0.9 µM and 4.5 µM, respectively. The standard error of the determinations (SEM) was ± 10% (n = 2). (C) ELISA experiment. The plate was coated with the human (●, bold line) and sponge (○, dashed line) TPPP forms, and then it was incubated with tubulin at different concentrations. The bound tubulin was detected by tubulin antibody. The binding afﬁnities of the TPPP forms to tubulin were determined by curve fitting using hyperbolic saturation and were found to be 38 ± 6 and 30 ± 0.6 nM for human and sponge TPPP, respectively. Error bars show the standard error of the determinations (SEM) (n = 2). Figure 5. Interaction of the human and sponge TPPP with tubulin. (A) Pelleting experiment. After the incubation of 5 µM tubulin with 10 µM of each TPPP form, the pellet (P) and the supernatant (S) fractions obtained by centrifugation were analyzed by SDS-PAGE on a 13.5% gel. Tub: tubulin, Hs1: human TPPP, Sd: Suberites domuncula TPPP, tr Sd: truncated, ∆129-180 S. domuncula TPPP. MM: molecular weight marker (170, 130, 100, 70, 55, 40, 35, 25, 15 and 10 kDa). (B) Turbidity measurement to study the tubulin polymerization promoting activity of the various TPPP forms. To induce microtubule polymerization, 3 µM of human TPPP1 (bold line) or 6 µM sponge TPPP (dashed line) or 12 µM truncated sponge TPPP (dotted line) was added to the solution of tubulin (6 µM), respectively. The standard error of the determinations (SEM) was ± 10% (n = 3). (C) Tubulin (12 µM) polymerization in the presence of paclitaxel (20 µM) and subsequently added (indicated by an arrow) 3 µM of human TPPP1 (bold line) or 6 µM of sponge (dashed line) TPPP.


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Figure 6. Electron microscopic analysis of the effect of TPPPs on tubulin polymerization. (A-B) Negatively stained whole mounts. (C-D) Sections from resin-embedded pellets. Microtubules and aggregates formed in the presence of Suberites domuncula TPPP (A, B and D) and human TPPP1 (C). TPPPs induce the formation of loosely arranged microtubule bundles as well. Bars: 500 nm. Figure 7. Electron microscopic analysis of the effect of TPPPs on paclitaxel-stabilized microtubules. Sections from resin-embedded pellets. Paclitaxel-stabilized microtubules (A) treated with human TPPP1 (B) or Suberites domuncula TPPP (C, D). (A) Loosely arranged, long straight, and curved microtubules formed in the absence of TPPPs. (B) TPPPs induce the formation of large microtubule bundles. Cross sections as well as longitudinal sections are visible. Bar: 500 nm.


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Figure 1 99x58mm (300 x 300 DPI)

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Figure 4 189x424mm (300 x 300 DPI)


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Figure 5 139x115mm (300 x 300 DPI)


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Figure 6 119x84mm (300 x 300 DPI)


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Figure 7 119x84mm (300 x 300 DPI)


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For Table of Contents Use Only Tubulin binding and polymeriza 34x14mm (300 x 300 DPI)


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