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The Hungarian Academy of Sciences (HAS, established in 1825), similar to the academies of the old Soviet bloc, ran a research network from 1950 until 2019 when it was detached from the Academy. The first research institute of the HAS was the Institute of Biochemistry, which started its operation in 1950. Its first director was Imre Szörényi (1905–1959) who lived in emigration in Kiev until he was called back to Hungary in 1950 by the Secretariat of the Hungarian Workers Party. Initially, for a few years research in the Institute was partly influenced by Lepeshinskaya's ‘New Cell Theory’ and Szörényi himself became the chair of the ‘Living Protein’ Committee of the HAS. He returned for more than two years to Kiev where he received a shared Stalin Prize in 1952 for the development of the antibiotic, Microcid. After his final return to Hungary in 1953, he was able to shape the characteristic image of the Institute of Biochemistry, making it one of the leading workshops of Hungarian biochemistry. From 1956 onwards, ideological considerations no longer interfered with the choice of research topics. The relationship between the chemical structure and the specific biological function of enzymes became the main profile of the Institute. In spite of his untimely death, Szörényi exerted a long-lasting influence on Hungarian biochemistry through his disciples.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s40656-021-00442-2.

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.

This paper highlights a general problem, namely that host genome sequences can easily be contaminated with parasite sequences, thus careful isolation of genetic material and careful bioinformatics analysis are needed in all cases. Two recently published genomes are shown here to be contaminated with sequences of apicomplexan parasites which belong to the Sarcocystidae family. Sequences of the characteristic apicomplexan organelle, the apicoplast, were used as queries in BLASTN searches against nucleotide sequences of various animal groups looking for possible contamination. Draft genomes of a bird, Colinus virginianus (Halley et al., 2014), and a bat, Myotis davidii (Zhang et al., 2013) were found to contain at least six and 17 contigs, respectively, originating from the apicoplast of an apicomplexan species, and other genes specific to this phylum can also be found in the published genomes. Obviously, the sources of the genetic material, the muscle and the kidney of the animals, respectively, contained the parasitic cysts. Phylogenetic analyses using 18S rRNA and internal transcribed spacer 1 genes show that the parasite contaminating C. virginianus is a species of Sarcocystis related to ones known to cycle between avian and mammalian hosts. In the case of M. davidii it belongs to the Nephroisospora genus, the only member of which, Nephroisospora eptesici, has been recently identified from the kidney of big brown bats (Eptesicus fuscus).

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.

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.

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.

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin, and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting tubulin polymerization promoting protein, TPPP/p25. TPPP/p25 is a microtubule-associated protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The nonphysiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed.

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.

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.

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 β-amyloid (Aβ) 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 Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ 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 Aβ42 tightly bound to TPPP/p25 (Kd = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ42, 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 Aβ 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 Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.

The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays a crucial role in differentiation of oligodendrocytes. Here we first demonstrated by multinuclear NMR that the extended disordered segments are localized at the N- and C-terminals straddling a flexible region. We showed by affinity chromatography, fluorescence spectroscopy and circular dichroism that GTP binds to TPPP/p25 likely within the flexible region; neither positions nor intensities of the peaks in the assigned terminals were affected by GTP. In addition, we demonstrated that TPPP/p25 specifically hydrolyses GTP in an Mg2+-dependent manner. The GTPase activity is comparable with the intrinsic activities of small G proteins suggesting its potential role in multiple physiological processes.

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.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered V(max) values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences.

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K(d) = 2.5-2.7 microM) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.

TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976-13981]. We identified two human gene sequences, CG1-38 and p25beta, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.

Bisindole Vinca alkaloids target microtubule system causing anti-mitotic activity. The problem of their clinical application is the lack of selectivity resulting in toxic side effects. In this paper we review the late history of new bisindole derivatives focusing on KARs recognized as potent anti-cancer drugs with low side effect. KARs, just as other bisindoles, impede microtubule assembly of mitotic spindle, however, they display no anti-calmodulin activity. This new drug family appears to be less potent than vinblastine in vitro systems, but it shows high antitumor efficacy with considerably higher doses being well tolerated in the animal tumor models. 3D data of calmodulin complexed with KAR-2 explain the specificity and unique pharmacology of KAR derivatives.

TPPP/p25, a flexible unstructured protein, binds to tubulin and induces aberrant microtubule assemblies. We identified hereby glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a new interacting partner of TPPP/p25. The immunoprecipitation and affinity chromatographic experiments with bovine brain cell-free extract revealed that the interaction was salt and NAD+ sensitive while ELISA showed resistant and firm association of the two isolated proteins. In transfected HeLa cells at low expression level of EGFP-TPPP/p25, while the green fusion protein aligned at the microtubular network, GAPDH distributed uniformly in the cytosol. However, at high expression level, GAPDH co-localized with TPPP/p25 in the aggresome-like aggregate. Immunohistochemistry showed enrichment of TPPP/p25 and GAPDH within the α-synuclein positive Lewy body.

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TPPP/p25, the first representative of a new protein family, identified as a brain-specific unfolded protein induces aberrant microtubule assemblies in vitro, suppresses mitosis in Drosophila embryo and is accumulated in inclusion bodies of human pathological brain tissues. In this paper, we present prediction and additional experimental data that validate TPPP/p25 to be a new member of the “intrinsically unstructured” protein family. The comparison of these characteristics with that of a-synuclein and tau, involved also in neurodegenerative diseases, suggested that although the primary sequences of these proteins are entirely different, there are similarities in their well-defined unstructured segments interrupted by “stabilization centres”, phosphorylation and tubulin binding motives. SK-N-MC neuroblastoma cells were transfected with pEGFP-TPPP/p25 construct and a stable clone denoted K4 was selected and used to establish the effect of this unstructured protein on the energy state/metabolism of the cells. Our data by analyzing the mitochondrial membrane polarization by fluorescence microscopy revealed that the high-energy phosphate production in K4 clone is not damaged by the TPPP/p25 expression. Biochemical analysis with cell homogenates provided quantitative data that the ATP level increased 1.5-fold and the activities of hexokinase, glucosephosphate isomerase, phosphofructokinase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase were 1.2 to 2.0-fold higher in K4 as compared to the control. Our modelling using these data and rate equations of the individual enzymes suggests that the TPPP/p25 expression stimulates glucose metabolism. At pathological conditions TPPP/p25 is localized in inclusion bodies in multiple system atrophy, it tightly co-localizes with a-synuclein, partially with tubulin and not with vimentin. The previous and the present studies obtained with immunohistochemistry with pathological human brain tissues rendered it possible to classify among pathological inclusions on the basis of immunolabelling of TPPP/p25, and suggest this protein to be a potential linkage between Parkinson’s and Alzheimer’s diseases. © 2004 Elsevier SAS. All rights reserved.

Recently, we isolated from bovine brain a protein, TPPP/p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via blast . Here, we characterized the binding of TPPP/p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: k on , 2.4 × 10 4 M –1 ·s –1 ; k off , 5.4 × 10 –3 s –1 ; and K d , 2.3 × 10 –7 M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP/p25 into cleavage Drosophila embryos expressing tubulin–GFP fusion protein reveals that TPPP/p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP/p25 both in vitro and in vivo .

Experimental and model studies were performed to characterize the flux of glucose metabolism and the sharing of glucose-6-phosphate (Glu6P) by the upper parts of glycolytic and pentosephosphate pathways in the brain extract. A mathematical model based upon the kinetic equations of the individual enzymes was evaluated to fit the experimental data. Glucose is converted to glucose-6-phosphate by hexokinase that controls almost exclusively the glucose metabolism. Experiments showed that this crossroad-metabolite was shared between glycolysis and pentosephosphate pathway in the brain extract in a ratio of 1.5:1. This ratio was favorable to the pentosephosphate pathway by the addition of high excess of exogenous glucose-6-phosphate dehydrogenase, standardly used for the activity assay of hexokinase, but still a significant part (17+/-3%) of the common intermediate was converted into the direction of glycolysis. Stimulation of glucose-6-phosphate formation via moderate (30-50%) increase of hexokinase activity by adding exogenous hexokinase or tubulin resulted in the slight increase of the relative flux into direction of glycolysis. The model correctly described all of these observations. However, when the activity of hexokinase was doubled with exogenous enzyme, significantly less glucose-6-phosphate was converted into direction of glycolysis than predicted. This discrepancy shows that the system did not behave in this case as an ideal one, which could be due to the formation of distinct pools for the intermediate.

Previously, we have described a linearization procedure for the determination of dissociation constants of antigen-antibody interactions using data from enzyme-linked immunosorbent assays (ELISA) [J. Immunol. Methods 143 (1991) 119]. Here, we present a new linearization procedure-based partly on the former one-to evaluate quantitatively the data of displacement ELISAs. It renders possible the determination of dissociation constants of complexes formed between immobilised antigens and the displacing molecules without knowing the explicit concentration of the antigen and the dissociation constant and stoichiometry of the immunocomplex. In addition, determination of the concentration of specific antibody in the serum is not necessary since the equations use only the ratio of serum concentration and apparent dissociation constant of the immunocomplex (both of which can be expressed as serum dilutions). The method has been applied to determine the strength of immobilised hexokinase-tubulin interactions (K(d)=1.2 microM) from a displacement ELISA using antihexokinase serum. The applicability and limitations of the procedure are discussed on the basis of both the experimental and theoretical data.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC50 = 4.0 ˛ 1.4 WM). This value virtually was not affected by specific ligands such as ATP or glucose 6- phosphate. Microtubule-bound hexokinase obtained in reconsti- tuted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed. fl 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

In a Hungarian family with triosephosphate isomerase (TPI; d -glyceraldehyde-3-phosphate keto-isomerase, EC 5.3.1.1 ) deficiency, two germ-line identical, but phenotypically differing compound heterozygote brothers (one of them with neurological disorder) have been identified with the same very low ( brother without neurological disorder > normal control. This distinct microcompartmentation of mutant proteins may be relevant in the development of the neurodegenerative process in TPI deficiency and in other, more common neurological diseases.

The numerous antimitotic drugs already developed and therapeu- tically used for a large number of pathologies belong to different classes based on their chemical structure and other distinctive features. Antimitotic drugs usually target the tubulin/microtubule network of the cytoskeleton that is formed by assembly of cyto- plasmic tubulin dimers. The stability of the different microtubular structures varies considerably; some of their functions depend on Summary A new semisynthetic anti-tumour bis-indol compound, KAR-2 (3'-(β-chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxy- vinblastine) with lower toxicity than vinca alkaloids used in chemotherapy binds to calmodulin but, in contrast to vinblastine, does not exhibit anti-calmodulin activity. To investigate whether the modest chemical modification of bis-indol structure is responsible for the lack of anti- calmodulin potency and for the different pharmacological effects, new derivatives have been synthesized for comparative studies. The synthesis of the KAR derivatives are presented. The comparative studies showed that the spiro-oxazolidino ring and the substitution of a formyl group to a methyl one were responsible for the lack of anti-calmodulin activities. The new derivatives, similar to the mother compounds, inhibited the tubulin assembly in polymerization tests in vitro, however their inhibitory effect was highly dependent on the organization state of microtubules; bundled microtubules appeared to be resistant against the drugs. The maximal cytotoxic activities of KAR derivatives in in vivo mice hosting leukaemia P388 or Ehrlich ascites tumour cells appeared similar to that of vinblastine or vincristine, however significant prolongation of life span could be reached with KAR derivatives only after the administration of a single dose. These studies plus data obtained using a cultured human neuroblastoma cell line showed that KAR compounds displayed their cytotoxic activities at significantly higher concentrations than the mother compounds, although their antimicrotubular activities were similar in vitro. These data suggest that vinblastine/vincristine damage additional crucial cell functions, one of which could be related to calmodulin-mediated processes.

Simultaneous binding of two sequential glycolytic enzymes, phosphofructokinase and aldolase, to a microtubular network was investigated. The binding of the phosphofructokinase to microtubules and its bundling activity has been previously characterized (Lehotzky, A., Telegdi, M., Liliom, K., and Ovadi, J. (1993) J. Biol. Chem. 268, 10888-10894). Aldolase binding to microtubules at near physiological ionic strength is weak (Kd = 20 microM) as compared with that of the kinase (Kd = 1 microM). The interactions of both enzymes with microtubules are modulated by their common intermediate, fructose-1,6-bisphosphate. Pelleting and electron microscopic measurements have revealed that the aldolase binding interferes with that of phosphofructokinase, although they have distinct binding domains on microtubules. The underlying molecular mechanism responsible for this finding is that in the solution phase aldolase and phosphofructokinase form a bienzyme complex that does not bind to the microtubule. The bienzyme complex formation does not influence the catalytic activity of aldolase, however, it inhibits the dissociation-induced inactivation of the kinase by stabilizing a catalytically active molecular form. The present data suggest the first experimental evidence that two sequential glycolytic enzymes do not associate simultaneously to microtubules, but their complexation in solution provides kinetic advantage for glycolysis.

KAR-2 (3′′-(β-chloroethyl)-2′′,4′′-dioxo-3,5′′-spiro-oxazolidino-4-deacetoxy-vinblastine) is a semisynthetic bis-indol derivative, with high anti-microtubular and anti-tumour activities but with low toxicity. KAR-2, in contrast to other biologically active bis-indols (e.g. vinblastine), did not show anti-calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR-2 to calmodulin. The binding affinity of KAR-2 to calmodulin (dissociation constant was about 5 μM) in the presence of Ca2+ was comparable to that of vinblastine. KAR-2 was able to interact with apo-calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR-2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady-state level. Comparison of the data obtained with KAR-2 and bis-indols used in chemotherapy suggests that the lack of anti-calmodulin potency resides on the spiro-oxazolidino portion of KAR-2. This character of KAR-2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti-tumour test.

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5"-spiro-oxazolidino- 4-deacetoxy-vinblastine), is a bis-indol derivative; catharantine is coupled with the vindoline moiety which contains a substituted oxazolidino group. Our binding studies showed that KAR-2 exhibited high affinity for bovine purified brain tubulin (Kd-3 microM) and it inhibited microtubule assembly at a concentration of 10 nM. 2. Anti-microtubular activity of KAR-2 was highly dependent on the ultrastructure of microtubules: while the single tubules were sensitive, the tubules cross-linked by phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) exhibited significant resistance against KAR-2. 3. The cytoplasmic microtubules of Chinese hamster ovary mammalian and Sf9 insect cells were damaged by 1 microgram ml-1 KAR-2, as observed by indirect immunofluorescence and transmission electron microscopy. Scanning electron microscopy revealed intensive surface blebbing on both types of cells in the presence of KAR-2. 4. KAR-2 was effective in the mouse leukaemia P338 test in vivo without significant toxicity. Studies on a primary cerebro-cortical culture of rat brain and differentiated PC12 cells indicated that the toxicity of KAR-2 was significantly lower than that of vinblastine. The additional property of KAR-2 that distinguishes it from bis-indol derivatives is the lack of anti-calmodulin activity.

article Background: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dy- namics 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 mul- tiple equilibrium systems from ITC measurements. Methods: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular di- chroism, 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 ad- dition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by di- sulfide 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 mathe- matical 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 poten- cy is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the un- folded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.

A new protein, termed apicortin, has recently been identified, which occurs only in the placozoan animal Trichoplax adhaerens and in the genomes of all currently sequenced apicomplexan parasites (e.g., Toxoplasma, Plasmodium, etc.). Apicortins unite two conserved domains, a DCX motif and a partial p25alpha sequence, which are singly found in diverse other proteins, in doublecortins and TPPPs, respectively. Here I show that although apicortin has a limited phylogenomic distribution, its occurrence is broader than thought previously. It has been identified in another primitive opisthokont, in the chytrid fungus, Spizellomyces punctatus. Apicortins can be divided into two subgroups: apicomplexan and non-apicomplexan ones. The main difference is that the former ones possess a long, N-terminal extension predicted to be disordered, which is missing in the other subgroup. The appearance of the extension in apicomplexan apicortins is an “innovation” of this phylum, and may play a functional role in the protein–protein interactions.

In our experiment we tried to find out what kind of eventual changes in the environment and in plant chemical composition occurred in response to different fertilizer treatments applied to sweet corn (Zea mays convar. saccharata) grown on sandy soil with low humus content. The ploughed layer contained1% CaCO3 and around 1% humus. The soil was very well supplied with P, well supplied with K, Mg, Mn and Cu, and weakly supplied with N and Ca. The treatments were planned in accordance with the recommendations, with a planned unhusked ear yield of 16 tons per hectare, of the new environmental friendly advisory system recently elaborated for field vegetable crops in Hungary. The treatments applied included: G1 (blank control)(N0P0K0), G2(N222.5P22.2K143), G3(N445 P22.5 K143), G4(N222.5 P22.5K143), G5(N222.5P22.5 K286), G6(N222.5 P22.5 K143) + Mg(1.52). According to our findings, of the composition parameters of the grains of the treatments with no fertilizer application, the invert and reducing sugar contents (4.42%, respectively 2.59% relative to fresh weight(-1)) in grains were the highest among the treatments. The same conclusion was drawn on the K 120.2, Mg 13.3, Fe 0.24, Cu 0.66 mg 100 g(-1) grain dry weight levels among minerals. In the case of the basic treatment (G2) recommended by the advisory system we obtained favourable results for the measured parameters, including yields. Invert and reducing sugar contents were (3.26% respectively 1.97% relative to fresh weight(-1)), and mineral contents K 101.9; Mg 11.8; Fe 0.21; Cu 0.56 mg 100 g(-1) dry weight. In the grains, no translocation of toxic elements was observed in response to the direct or indirect effect of the treatments.

A new protein, termed apicortin, has been identified, which contains a DCX (doublecortin) and a partial p25-alpha domain. The DCX domains of the doublecortin superfamily are responsible for their microtubule binding and stabilizing properties. The p25-alpha domain occurs in TPPPs (Tubulin Polymerization Promoting Proteins) exhibiting Microtubule Associated Protein (MAP)-like functions. TPPP orthologs can be classified as long- and short-type ones. The latter ones do not contain the partial p25-alpha domain, which is the most conservative part of the long-type TPPPs and was evolutionary-preserved independently from the whole domain. Apicortin seems to occur only in the placozoan animal Trichoplax adhaerens and in each of the apicomplexan parasites, the genomes of which are available. The function of the novel protein can be predicted from the fact that it contains two different microtubule-binding domains. Apicomplexans, important pathogens of humans and animals, possess unique cytoskeletal elements, as subpellicular microtubules and apical polar rings, which are specific for this phylum, extremely stabilized by unknown MAPs. One of the candidates can be the apicortin, which is supported by a recent experimental proteomics analysis. The role of the new protein in T. adhaerens may be similar but needs clarification.

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.