The Role of Tubulin Polymerization-Promoting Protein2 (TPPP2) in Spermatogenesis: A Narrative Review.

Tubulin polymerization-promoting protein2 (TPPP2) is one of the three paralogs of mammalian TPPP proteins. Its possible role in spermatogenesis is described in this narrative review. TPPP2 is expressed specifically in the male reproductive system, mainly in testes and sperm, and also in the epididymis. In testes, TPPP2 is exclusively expressed in elongating spermatids; in the epididymis, it is located in the middle piece of the sperm tail. TPPP2 is involved in spermiogenesis, in steps which are determinative for the formation and morphology of spermatids. The inhibition of TPPP2 decreases sperm motility (the curvilinear velocity of sperms), probably due to influencing mitochondrial energy production since TPPP2 knockout mice possess an impaired mitochondrial structure. There are data on the role of TPPP2 in various mammalian species: human, mouse, swine, and various ruminants; there is a significant homology among TPPP2s from different species. Experiments with Tppp2-/--mice show that the absence of TPPP2 results in decreased sperm count and serious dysfunction of sperm, including decreased motility; however, the in vitro capacitation and acrosome reaction are not influenced. The symptoms show that Tppp2-/--mice may be considered as a model for oligoasthenozoospermia.

On the TPPP Protein of the Enigmatic Fungus, Olpidium-Correlation between the Incidence of p25alpha Domain and That of the Eukaryotic Flagellum.

Loss of the flagellum was an important step in the evolution of fungi. The flagellated fungi of the phylum Olpidiomycota are the closest relative of the non-flagellated terrestrial fungi. There are genes encoding proteins, the occurrence of which shows a strong correlation with the incidence of the flagellum. One of these gene/protein families is "TPPP-like proteins" whose main feature is the presence of the p25alpha domain. The functional link between TPPP and flagellum has also been shown. Most of the phyla of flagellated fungi have been known to contain TPPP-like proteins but Olpidiomycota was an exception. This study demonstrates that Olpidium bornovanus, similarly to some fungi of Chytridiomycota and Blastocladiomycota, has a "fungal-type" TPPP characterized by the presence of two (a complete and an incomplete) p25alpha domains.

What's in a name? From "fluctuation fit" to "conformational selection": rediscovery of a concept.

Rediscoveries are not rare in biology. A recent example is the re-birth of the "fluctuation fit" concept developed by F. B. Straub and G. Szabolcsi in the sixties of the last century, under various names, the most popular of which is the "conformational selection". This theory offers an alternative to the "induced fit" concept by Koshland for the interpretation of the mechanism of protein-ligand interactions. A central question is whether the ligand induces a conformational change (as described by the induced fit model) or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of various states of the protein (according to the fluctuation fit/conformational selection model). Straub and Szabolcsi's role and the factors hindering the spread of the fluctuation fit theory are discussed in the context of the history of the Hungarian biology in the 1950s and 1960s.

Tubulin Binding and Polymerization Promoting Properties of Tubulin Polymerization Promoting Proteins Are Evolutionarily Conserved.

Tubulin polymerization promoting proteins (TPPPs) constitute a eukaryotic protein family. There are three TPPP paralogs in the human genome, denoted as TPPP1-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 whole-genome duplication; thus, whether the tubulin/microtubule binding function of TPPP1 and TPPP3 is a newly acquired property or was present in the invertebrate orthologs (generally one TPPP per species) has been an open question. 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 the sponge protein correspond to those of TPPP1; namely, it is an IUP that strongly binds tubulin and induces its polymerization, proving that these features of animal TPPPs have been evolutionarily conserved.

Wider than Thought Phylogenetic Occurrence of Apicortin, A Characteristic Protein of Apicomplexan Parasites.

Apicomplexan parasites cause serious illnesses, including malaria, in humans and domestic animals. The presence of apicortins is predominantly characteristic of this phylum. All the apicomplexan species sequenced contain an apicortin which unites two conserved domains: DCX and partial p25alpha. This paper identifies novel apicortin orthologs in silico and corrects in several cases the erroneous sequences of hypothetical apicortin proteins of Cryptosporidium, Eimeria, and Theileria genera published in databases. Plasmodium apicortins, except from Plasmodium gallinaceum, differ significantly from the other apicomplexan apicortins. The feature of this ortholog suggests that only orthologs of Plasmodiums hosted by mammals altered significantly. The free-living Chromerida, Chromera velia, and Vitrella brassicaformis, contain three paralogs. Their apicomplexan-type and nonapicomplexan-type apicortins might be "outparalogs." The fungal ortholog, Rozella allomycis, found at protein level, and the algal Nitella mirabilis, found as Transcriptome Shotgun Assembly (TSA), are similar to the known Opisthokont (Trichoplax adhaerens, Spizellomyces punctatus) and Viridiplantae (Nicotiana tabacum) ones, since they do not contain the long, unstructured N-terminal part present in apicomplexan apicortins. A few eumetazoan animals possess apicortin-like (partial) sequences at TSA level, which may be either contaminations or the result of horizontal gene transfer; in some cases the contamination has been proved.

On the tubulin polymerization promoting proteins of zebrafish.

Recently, Aoki et al. [15] have been published a paper (Biochem. Biophys. Res. Commun. 445 (2014) 357-362.) in which they identified possible downstream genes required for the extension of peripheral axons in primary sensory neurons of zebrafish. Tppp was claimed as one of them but, as I show, it is the tppp3-like gene, a paralog of tppp, which plays this role. There are three tppp paralogs in fishes: tppp1 (named also tppp), tppp3 and tppp3-like. Tppp1 and tppp3 are the orthologs of the corresponding human genes, however, the classification of the third one is ambiguous. It is known that the genomes of the early vertebrate lineage underwent two complete genome duplications, which result in the presence of several paralogs in vertebrates. A teleost fish specific third whole genome duplication also occurred. Thus the tppp3-like gene can be either an ortholog of human TPPP2 or a fourth paralog (tppp4) absent in tetrapods but present in fishes; finally a tppp3a gene which can be originated from the third, fish specific, whole genome duplication. Comparing the sequences of vertebrate and recently available lamprey tppps I show that the tppp3-like gene is a TPPP2 ortholog. Synteny data are in accordance with this suggestion.

Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data.

BACKGROUND: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements. METHODS: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software. RESULTS: The dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP addition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by disulfide bond or chemical cross-linking. The GTP binding to the dimeric form (Kd-GTP=200 µM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathematical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements. CONCLUSIONS/GENERAL SIGNIFICANCE: The dimerization of TPPP/p25 with favorable physiological functional potency is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the unfolded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.

Tubulin Polymerization Promoting Proteins (TPPPs) of Aphelidiomycota: Correlation between the Incidence of p25alpha Domain and the Eukaryotic Flagellum.

The seven most early diverging lineages of the 18 phyla of fungi are the non-terrestrial fungi, which reproduce through motile flagellated zoospores. There are genes/proteins that are present only in organisms with flagellum or cilium. It was suggested that TPPP-like proteins (proteins containing at least one complete or partial p25alpha domain) are among them, and a correlation between the incidence of the p25alpha domain and the eukaryotic flagellum was hypothesized. Of the seven phyla of flagellated fungi, six have been known to contain TPPP-like proteins. Aphelidiomycota, one of the early-branching phyla, has some species (e.g., Paraphelidium tribonematis) that retain the flagellum, whereas the Amoeboaphelidium genus has lost the flagellum. The first two Aphelidiomycota genomes (Amoeboaphelidium protococcorum and Amoeboaphelidium occidentale) were sequenced and published last year. A BLASTP search revealed that A. occidentale does not have a TPPP, but A. protococcorum, which possesses pseudocilium, does have a TPPP. This TPPP is the 'long-type' which occurs mostly in animals as well as other Opisthokonta. P. tribonematis has a 'fungal-type' TPPP, which is found only in some flagellated fungi. These data on Aphelidiomycota TPPP proteins strengthen the correlation between the incidence of p25alpha domain-containing proteins and that of the eukaryotic flagellum/cilium.

Presence of p25alpha-Domain in Seed Plants (Spermatophyta): Microbial/Animal Contaminations and/or Orthologs.

Genome and transcriptome assembly data often contain DNA and RNA contaminations from external organisms, introduced during nucleotide extraction or sequencing. In this study, contamination of seed plant (Spermatophyta) transcriptomes/genomes with p25alpha domain encoding RNA/DNA was systematically investigated. This domain only occurs in organisms possessing a eukaryotic flagellum (cilium), which seed plants usually do not have. Nucleotide sequences available at the National Center for Biotechnology Information website, including transcriptome shotgun assemblies (TSAs), whole-genome shotgun contigs (WGSs), and expressed sequence tags (ESTs), were searched for sequences containing a p25alpha domain in Spermatophyta. Despite the lack of proteins containing the p25alpha domain, such fragments or complete mRNAs in some EST and TSA databases were found. A phylogenetic analysis showed that these were contaminations whose possible sources were microorganisms (flagellated fungi, protists) and arthropods/worms; however, there were cases where it cannot be excluded that the sequences found were genuine hits and not of external origin.

The Unicellular, Parasitic Fungi, Sanchytriomycota, Possess a DNA Sequence Possibly Encoding a Long Tubulin Polymerization Promoting Protein (TPPP) but Not a Fungal-Type One.

The unicellular, parasitic fungi of the phylum Sanchytriomycota (sanchytrids) were discovered a few years ago. These unusual chytrid-like fungi parasitize algae. The zoospores of the species of the phylum contain an extremely long kinetosome composed of microtubular singlets or doublets and a non-motile pseudocilium (i.e., a reduced posterior flagellum). Fungi provide an ideal opportunity to test and confirm the correlation between the occurrence of flagellar proteins (the ciliome) and that of the eukaryotic cilium/flagellum since the flagellum occurs in the early-branching phyla and not in terrestrial fungi. Tubulin polymerization promoting protein (TPPP)-like proteins, which contain a p25alpha domain, were also suggested to belong to the ciliome and are present in flagellated fungi. Although sanchytrids have lost many of the flagellar proteins, here it is shown that they possess a DNA sequence possibly encoding long (animal-type) TPPP, but not the fungal-type one characteristic of chytrid fungi. Phylogenetic analysis of p25alpha domains placed sanchytrids into a sister position to Blastocladiomycota, similarly to species phylogeny, with maximal support.

p25alpha Domain-Containing Proteins of Apicomplexans and Related Taxa.

TPPP (tubulin polymerization promoting protein)-like proteins contain one or more p25alpha (Pfam05517) domains. TPPP-like proteins occur in different types as determined by their length (e.g., long-, short-, truncated-, and fungal-type TPPP) and include the protein apicortin, which possesses another domain, doublecortin (DCX, Pfam 03607). These various TPPP-like proteins are found in various phylogenomic groups. In particular, short-type TPPPs and apicortin are well-represented in the Myzozoa, which include apicomplexans and related taxa, chrompodellids, dinoflagellates, and perkinsids. The long-, truncated-, and fungal-type TPPPs are not found in the myzozoans. Apicortins are found in all apicomplexans except one piroplasmid species, present in several other myzozoans, and seem to be correlated with the conoid and apical complex. Short-type TPPPs are predominantly found in myzozoans that have flagella, suggesting a role in flagellum assembly or structure.

Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein.

The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with ß-amyloid (Aß) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aß oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aß with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aß(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aß(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aß was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aß can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aß and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.

A fish-specific member of the TPPP protein family?

A eukaryotic protein family, the tubulin polymerization promoting proteins (TPPPs), has recently been identified. It has been termed after its first member, TPPP/p25 or TPPP1, which exhibits microtubule-stabilizing function and plays a role in neurodegenerative diseases. In mammalian genomes, two further paralogues, TPPP2 and TPPP3, can be found. In this article, I show that TPPP1 and TPPP3, but not TPPP2, are included in paralogons, on human chromosomes, Hsa5 and Hsa16, respectively. I suggest that the single non-vertebrate tppp gene was duplicated in the first round of whole-genome duplication in the vertebrate lineage giving rise to tppp1 and the precursor of tppp2/tppp3. The existence of a teleost fish-specific fourth paralogue, tppp4, has also been raised, but it is not supported by synteny analysis. Alternatively, the new group can be considered as the fish orthologue of TPPP2. The case that the new group is the consequence of the teleost fish-specific whole-genome duplication (3R) cannot be excluded.

Proteins without 3D structure: definition, detection and beyond.

MOTIVATION: Predictions, and experiments to a lesser extent, following the decoding of the human genome showed that a significant fraction of gene products do not have well-defined 3D structures. While the presence of structured domains traditionally suggested function, it was not clear what the absence of structure implied. These and many other findings initiated the extensive theoretical and experimental research into these types of proteins, commonly known as intrinsically disordered proteins (IDPs). Crucial to understanding IDPs is the evaluation of structural predictors based on different principles and trained on various datasets, which is currently the subject of active research. The view is emerging that structural disorder can be considered as a separate structural category and not simply as absence of secondary and/or tertiary structure. IDPs perform essential functions and their improper functioning is responsible for human diseases such as neurodegenerative disorders.

An unstructured protein with destructive potential: TPPP/p25 in neurodegeneration.

TPPP/p25 is a recently discovered, unstructured protein involved in brain function. It is found predominantly in oligodendrocytes in normal brain but is enriched in neuronal and glial inclusions of Parkinson's disease and other synucleinopathies. Its physiological function seems to be the dynamic stabilization of microtubular ultrastructures, as well as the projections of mature oligodendrocytes and ciliary structures. We reappraise the earlier belief that TPPP/p25 is a brain-specific protein. We have identified and cloned two shorter (N-terminal-free) homologs of TPPP/p25 that behave differently from each other and from TPPP/p25. Two unique cell models have been established and used to study the effect of the unstructured protein on the energy metabolism and the formation of pathological aggregates. Our data suggest that the intracellular level of TPPP/p25 influences the cell differentiation, proliferation and the formation of protein aggregates, and consequently, the etiology of central nervous system diseases.

Apicortin, a Constituent of Apicomplexan Conoid/Apical Complex and Its Tentative Role in Pathogen-Host Interaction.

In 2009, apicortin was identified in silico as a characteristic protein of apicomplexans that also occurs in the placozoa, Trichoplax adhaerens. Since then, it has been found that apicortin also occurs in free-living cousins of apicomplexans (chromerids) and in flagellated fungi. It contains a partial p25-α domain and a doublecortin (DCX) domain, both of which have tubulin/microtubule binding properties. Apicortin has been studied experimentally in two very important apicomplexan pathogens, Toxoplasma gondii and Plasmodium falciparum. It is localized in the apical complex in both parasites. In T. gondii, apicortin plays a key role in shaping the structure of a special tubulin polymer, conoid. In both parasites, its absence or downregulation has been shown to impair pathogen-host interactions. Based on these facts, it has been suggested as a therapeutic target for treatment of malaria and toxoplasmosis.

Truncated TPPP - An Endopterygota-specific protein.

TPPP proteins exhibiting microtubule stabilizing function constitute a eukaryotic protein superfamily, characterized by the presence of the p25alpha domain of various lengths. Vertebrate species possess three TPPP paralogs; all of them possess a full-length p25alpha domain of 160-170 amino acids and are encoded by three exons. Species of Endopterygota (Holometabola) have, besides a full-size TPPP ortholog, a protein with a truncated p25alpha domain as well, where the last coding exon, responsible for microtubule binding, is missing. It is not the result of an alternative splicing but is coded by another gene. In Drosophila melanogaster, they are named as CG45057 (long-type) and CG6709 (truncated). The truncated protein has been found in the Endopterygota orders Diptera, Coleoptera, Hymenoptera, Lepidoptera and Raphidioptera. In Lepidoptera, in several superfamilies (Gelechioidea, Bombycoidea, Noctuoidea, Pyraloidea) two paralogs of the truncated TPPP occur. Truncated orthologs (CG6709) were not found in other insects or in arthropods and are absent in any other organism, as well, while the long-type TPPPs (CG45057 orthologs) occur commonly in all animals. Thus it seems that CG6709 orthologs occur only in insects undergoing on metamorphosis.

On the TPPP-like proteins of flagellated fungi.

TPPP-like proteins, exhibiting microtubule stabilizing function, constitute a eukaryotic superfamily, characterized by the presence of the p25alpha domain. TPPPs in the strict sense are present in animals except Trichoplax adhaerens, which instead contains apicortin where a part of the p25alpha domain is combined with a DCX domain. Apicortin is absent in other animals and occurs mostly in the protozoan phylum, Apicomplexa. A strong correlation between the occurrence of p25alpha domain and that of the eukaryotic cilium/flagellum was suggested. Species of the deeper branching clades of Fungi possess flagellum but others lost it thus investigation of fungal genomes can help testing of this suggestion. Indeed, these proteins are present in early branching Fungi. Both TPPP and apicortin are present in Rozellomycota (Cryptomycota) and Chytridiomycota, TPPP in Blastocladiomycota, apicortin in Neocallimastigomycota, Monoblepharomycota and the non-flagellated Mucoromycota. Beside the "normal" TPPP occurring in animals, a special, fungal-type TPPP is also present in Fungi, in which a part of the p25alpha domain is duplicated. Dikarya, the most developed subkingdom of Fungi, lacks both flagellum and TPPPs. Thus it is strengthened that each ciliated/flagellated organism contains p25alpha domain-containing proteins while there are very few non-flagellated ones where p25alpha domain can be found.

Triosephosphate isomerase deficiency: new insights into an enigmatic disease.

The triosephosphate isomerase (TPI) functions at a metabolic cross-road ensuring the rapid equilibration of the triosephosphates produced by aldolase in glycolysis, which is interconnected to lipid metabolism, to glycerol-3-phosphate shuttle and to the pentose phosphate pathway. The enzyme is a stable homodimer, which is catalytically active only in its dimeric form. TPI deficiency is an autosomal recessive multisystem genetic disease coupled with hemolytic anemia and neurological disorder frequently leading to death in early childhood. Various genetic mutations of this enzyme have been identified; the mutations result in decrease in the catalytic activity and/or the dissociation of the dimers into inactive monomers. The impairment of TPI activity apparently does not affect the energy metabolism at system level; however, it results in accumulation of dihydroxyacetone phosphate followed by its chemical conversion into the toxic methylglyoxal, leading to the formation of advanced glycation end products. By now, the research on this disease seems to enter a progressive stage by adapting new model systems such as Drosophila, yeast strains and TPI-deficient mouse, which have complemented the results obtained by prediction and experiments with recombinant proteins or erythrocytes, and added novel data concerning the complexity of the intracellular behavior of mutant TPIs. This paper reviews the recent studies on the structural and catalytic changes caused by mutation and/or nitrotyrosination of the isomerase leading to the formation of an aggregation-prone protein, a characteristic of conformational disorders.

Microtubule-Associated Proteins with Regulatory Functions by Day and Pathological Potency at Night.

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeleton, including the microtubule network. Microtubules play crucial roles achieved by their decoration with proteins/enzymes as well as by posttranslational modifications. This review focuses on the Tubulin Polymerization Promoting Protein (TPPP/p25), a new microtubule associated protein, on its "regulatory functions by day and pathological functions at night". Physiologically, the moonlighting TPPP/p25 modulates the dynamics and stability of the microtubule network by bundling microtubules and enhancing the tubulin acetylation due to the inhibition of tubulin deacetylases. The optimal endogenous TPPP/p25 level is crucial for its physiological functions, to the differentiation of oligodendrocytes, which are the major constituents of the myelin sheath. Pathologically, TPPP/p25 forms toxic oligomers/aggregates with α-synuclein in neurons and oligodendrocytes in Parkinson's disease and Multiple System Atrophy, respectively; and their complex is a potential therapeutic drug target. TPPP/p25-derived microtubule hyperacetylation counteracts uncontrolled cell division. All these issues reveal the anti-mitotic and α-synuclein aggregation-promoting potency of TPPP/p25, consistent with the finding that Parkinson's disease patients have reduced risk for certain cancers.