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3′-(β-Chloroethyl)-2′,4′-dioxo-3,5′-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca2+-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation (1H-15N heteronuclear single quantum coherence) spectra of 15N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.

Recently we identified TPPP/p25 (tubulin polymerization promoting protein/p25) as a brain-specific unstructured protein that induced aberrant microtubule assemblies and ultrastructure in vitro and as a new marker for Parkinson's disease and other synucleopathies. In this paper the structural and functional consequences of TPPP/p25 are characterized to elucidate the relationship between the in vitro and the pathological phenomena. We show that at low expression levels EGFP-TPPP/p25 specifically colocalizes with the microtubule network of HeLa and NRK cells. We found that the colocalization was dynamic (tg=5 seconds by fluorescence recovery after photobleaching) and changed during the phases of mitosis. Time-lapse and immunofluorescence experiments revealed that high levels of EGFP-TPPP/p25 inhibited cell division and promoted cell death. At high expression levels or in the presence of proteosome inhibitor, green fusion protein accumulated around centrosomes forming an aggresome-like structure protruding into the nucleus or a filamentous cage of microtubules surrounding the nucleus. These structures showed high resistance to vinblastin. We propose that a potential function of TPPP/p25 is the stabilization of physiological microtubular ultrastructures, however, its upregulation may directly or indirectly initiate the formation of aberrant protein aggregates such as pathological inclusions.

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.

The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186–200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinson's (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Pick's disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimer's disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

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.

Neomorphic moonlighting proteins perform distinct functions under physiological and pathological conditions without alterations at the gene level. The disordered tubulin-polymerization-promoting protein (TPPP/p25), a prototype of neomorphic moonlighting proteins, modulates the dynamics and stability of the microtubule system via its bundling and tubulin acetylation-promoting activities. These physiological functions are mediated by its direct associations with tubulin/microtubules as well as tubulin deacetylases such as histone deacetylase (HDAC) 6. In a normal brain, TPPP/p25 is expressed in oligodendrocytes and plays a crucial role in the formation of projections in the course of differentiation required for axon ensheathment. Under pathological conditions, TPPP/p25 interacts with α-synuclein, an aberrant protein–protein interaction resulting in aggregation leading to the formation of inclusions as clinical symptoms. The co-enrichment and co-localization of TPPP/p25 and α-synuclein were established in human-brain inclusions characteristic of Parkinson's disease (PD) and other synucleinopathies. The binding segments on TPPP/p25 involved in the physiological and the pathological interactions were identified and validated at molecular and cellular levels using recombinant proteins and transfected HeLa and inducible Chinese-hamster ovary (CHO) 10 cells expressing TPPP/p25. Our finding that distinct motifs are responsible for the neomorphic moonlighting feature of TPPP/p25, has powerful innovative effects in anti-Parkinson's disease drug research. Abbreviations: CDK5, cyclin-dependent kinase 5; CNS, central nervous system; ERK2, extracellular-signal-regulated kinase 2; HDAC, histone deacetylase; MAP, microtubule-associated protein; MS, multiple sclerosis; MSA, multiple system atrophy; PD, Parkinson’s disease; TPPP/p25, tubulin-polymerization-promoting protein

The highly dynamic cytoskeleton interacts with enzymes and other proteins that are involved in metabolic or signaling pathways. These interactions can influence the structural and functional characteristics of the partners at the microscopic level of individual proteins and polymers. In this work the functional consequences of such interactions have been studied at the macroscopic level in order to evaluate the integrative and regulatory roles of the metabolic pathways associated with the microtubule cytoskeleton. Here we present mathematical models of the interactions between a hypothetical metabolic pathway and microtubule assembly, and explore for the first time the functional consequences of these interactions in distinct situations. The models include kinetic constants of the individual steps and testable, relevant parameters which allow the quantification of the coupled processes at the microscopic and macroscopic levels. For example our kinetic model for the self-assembly of microtubules reproduces the alteration of the time-dependent turbidity caused by pyruvate kinase binding. Our data reveal the power of a mechanistic description of a filamentous system to explain how cells sense the state of metabolic and other pathways.

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.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

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.

Substrate channeling is the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing with the bulk phase. This process is equivalent to a microcompartmentation of the intermediate, although classic diffusion occurs simultaneously to varying extents in many of these cases. This microcompartmentation and other factors of channeling provide many potential biological advantages. Extensive examples of channeling can be found in the cited reviews. The choice of methods to detect and characterize substrate channeling depends extensively on the type of enzyme associations involved, the constants of the system, and, to some extent, the mechanism of channeling. Thus it is important to distinguish stable, dynamic, and catalytically induced enzyme associations as well as recognize different mechanisms of substrate channeling. We discuss the principles, experimental details, and limitations and precautions of five rather general methods. These use measurements of transient times, isotope dilution or enhancement, competing reaction effects, enzyme buffering kinetics, and transient-state kinetics. These encompass methods applicable to studies in vitro, in situ, and in vivo. None of these methods is applicable to all systems. They are also susceptible to artifacts without proper attention to precautions. Transient-state kinetic methods clearly excel in elucidating molecular mechanisms of channeling. However, they are often not the best method for initial detection and characterization of the process and they are not applicable to many complex systems. Several other methods that have been successful in indicating substrate channeling are briefly described.

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.

Experimental and model studies have been performed to characterise the effects of Cu2+ on the activities of individual glycolytic enzymes and on the flux and internal metabolite concentrations of the upper part of glycolysis in mouse muscle extracts. Cu2+ significantly inhibited the triosephosphate production from glucose with an IC50 of about 6.0 microM. At a similar extension Cu2+ inhibited hexokinase and phosphofructokinase, with an IC50 of 6.2 microM and 6.4 microM respectively, whereas the effects on the activities of aldolase, phosphoglucose isomerase and the internal metabolite levels were not significant. Flux control coefficients and flux response coefficients were determined in the presence of copper concentrations between 0 and 10 microM. The same values of flux control coefficients for hexokinase and for phosphofructokinase (0.8 and 0.2 respectively) were found in absence and in presence of copper. At Cu2+ equal to the flux IC50, the response coefficient was -1. The elasticity coefficients for hexokinase and phosphofructokinase at Cu2+ equal to the IC50 were also -1. A mathematical model was used to analyze the effect of copper on glycolysis under different conditions using experimental kinetic parameters and rate equations for enzymatic reactions of the upper part of glycolysis.

We identified the nonallosteric phosphofructokinase from the slime mold Dictyostelium discoideum as a potent protein factor that inhibits the rate of polymerization of tubulin at a molar ratio of 1 molecule to about 300 tubulin dimers for half-maximal action (IC50 = 32 nM). This effect was (i) assessed by turbidity measurements, pelleting of microtubules, and electron microscopy, (ii) observed when tubulin assembly was induced by taxol as well as by GTP in the presence of microtubule-associated proteins or glutamate, and (iii) specific as it was not produced by the phosphofructokinase from rabbit muscle. Also in contrast to the latter, neither tubulin nor microtubules modified the catalytic activity of the slime mold isozyme. Immunoelectron microscopy provided further evidence that D. discoideumphosphofructokinase physically interacts with tubulin, leading to the formation of aggregates. The process seems to be reversible since microtubules eventually formed in the presence of the inhibitor with concomitant reduction of tubulin aggregates. Limited proteolysis by subtilisin showed that the hypervariable C-termini of tubulin is not involved in the interaction with the enzyme. The possible physiological relevance of this novel function of D. discoideum phosphofructokinase different from its glycolytic action is discussed.

In normal rat and human brain tubulin, polymerization-promoting protein TPPP/p25 is predominantly localized in oligodendrocytes (Kovacs et al. in Neurobiol Dis 17:155–162, 2004; Lindersson et al. in J Biol Chem 280:5703–5715, 2005; Skjoerringe et al. in J Neurochem 99:333–342, 2006). TPPP/p25 promotes the polymerization of tubulin into double walled tubules and polymorphic aggregates and inhibits mitotic spindle assembly in vitro; furthermore, it is a common marker of -synuclein bearing pathological inclusions (summarized in detail: Kovacs et al., Acta Neuropath, this issue). Oligodendroglioma, oligoastrocytoma, clear cell ependymoma, and central neurocytoma share a common feature: the clear cell histopathology (Kleihues and Cavenee, IARC Press, Lyon, 2000). Therefore, diVerential diagnosis of clear cell primary brain tumours in the diagnostic setting often requires immunohistochemistry (Koperek et al. in Acta Neuropathol 108:24–30, 2004; Preusser et al. in Histopathology, in press). In this study, we evaluated the reliability of TPPP/p25 as an immunohistochemical marker of oligodendroglial neoplasms in the classiWcation and neuropathological diagnosis of primary clear cell brain tumours. Our cohort of human clear cell CNS neoplasms comprised 57 oligodendroglial neoplasms with chromosome 1p aberrations (1p deletion or imbalance status in 20 oligodendrogliomas, 20 anaplastic oligodendrogliomas, 17 oligoastrocytomas; 51/57 of these tumours additionally had aberrations of chromosome 19q), 3 oligodendroglial neoplasms without chromosome 1p aberration (2 oligodendrogliomas, 1 oligoastrocytoma), 10 central neurocytomas, 10 clear cell ependymomas, and 8 dysembryoplastic neuroepithelial tumours (DNTs). Further, we investigated 20 diVuse astrocytomas and glioblastomas, as well as 5 glioblastomas with clear cell components. Tumour typing was performed according to WHO criteria (Kleihues and Cavenee, IARC Press, Lyon, 2000). Aberrations of chromosome 1p and 19q were assessed by Xuorescence in situ hybridization (FISH) using the paracentromeric probe D1Z1 (1q12) and the subtelomeric probe D1Z2 (1p36.3; both Q-BIOgene, Heidelberg, Germany) for 1p and probes 19pter and 19q13.3 (both Vysis, BergishGladbach, Germany) for 19q as described in a previous study by our group (Gelpi et al. in Mod Pathol 16:708– 715, 2003). We also immunostained ten biopsy samples of non-neoplastic temporal cortex from patients diagnosed with chronic temporal lobe epilepsy. For immunohistochemistry, tissue specimens were Wxed in a phosphate-buVered 4.5% solution of formaldehyde, embedded in paraYn and cut at a thickness of 3–5 m. Sections were deparaVinized in xylene. Slides underwent heat induced epitope retrieval in 0.01 M citrate buVer (pH 6.0) in a steamer for 10 min. For antiTPPP/p25 immunostaining, we used two rat polyclonal M. Preusser · H. Budka · G. G. Kovacs Institute of Neurology, Medical University of Vienna, Vienna, Austria

Experimental and model studies have been performed to characterize the control properties of hexokinase and phosphofructokinase in muscle glycolysis and to examine the nature of error associated with experimental flux control coefficient determinations. Different approaches of metabolic control analysis, classical titration, co-response analysis and kinetic modelling indicated that flux control coefficients could be reliably estimated experimentally for the upper part of glycolysis. The kinetic parameters applied to construct the mathematical model were determined in muscle extract under similar conditions used for flux studies. If the kinetic parameters of commercial enzymes are introduced into the model the control analysis data cannot be trusted. Co-response analysis can also be successfully applied to determination of the flux control coefficients of the system. However, the involvement of a rapid-equilibrium enzyme, such as glucose 6-phosphate isomerase, could result in estimation errors for the relevant co-response coefficients that are propagated into the elasticity matrix. If the co-response coefficients related to isomerase activity are replaced by the values obtained by kinetic modelling, the values of elasticities are correct. Our data also suggest that in the upper part of glycolysis hexokinase mainly controls the pathway flux whereas phosphofructokinase exerts dominant control on the turnover of internal metabolite stocks inside the system.

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.

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.

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.

Cells face the enormous challenge of generating a single phenotype that must be coherent with myriad internal and external conditions. For such phenotypes to have multifarious but meaningful outputs entails the sensing, and integration of a wide variety of chemical and physical information, hence the coordination of metabolic and signaling processes. This sensing, integration, and coordination are carried out by the complex ultrastructural arrays and moonlighting functions of the cytoskeletal network. In the cellular context, the direction and potency of sensing are determined by the structure-related responses of the cytoskeletal network to the activity of individual macromolecules in conjunction with associated metabolites and nucleotides. These responses comprise the binding (hetero-association) of these macromolecules to the cytoskeleton and the consequences of this binding on the behavior of both partners, among them the stability and dynamics of the cytoskeleton, and the catalytic and regulatory properties of the individual proteins (and/or their specific complexes). The latter is of specific importance in regulation at a high level of organization via the formation of microcompartments in linear pathways or at metabolic crossroads. In addition, key players in many metabolic and signaling pathways are nucleotides such as ATP and GTP that have a crucial role in cytoskeleton-mediated events. These issues are illustrated with examples, and the sensing power of dynamic macromolecular associations is discussed.

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined.

The fact that enzyme complexes, stable, quasi-stable, and dynamic, exist in cells can no longer be ignored. Experimental evidence done with a variety of techniques has demonstrated these interactions in vitro and in vivo. There is scarcely a single known metabolic pathway in which no interactions of any of its enzymes exist (see reference 27 for a list of these). Such interactions are not only ubiquitous throughout metabolism, but they exist in all cell types, procaryote and eucaryote. In many of these systems the advantages of and regulatory power of enzyme-enzyme or enzyme-structural protein interactions has been amply demonstrated. The more difficult task is to assess accurately quantitative aspects of a system that varies between the solid, gel, and aqueous state. It is clear that the metabolic paradigm of soluble randomly dispersed metabolic elements in cells must be replaced, and new theoretical and experimental approaches introduced into this vital area of biological research.

The interaction of phosphofructokinase and microtubules results in mutual effects: decreases overall activity of the kinase and alters the ultrastructural organization of microtubules. Electron microscopic studies provide direct evidence for the periodical cross-bridges of microtubules by the kinase. 3-4 closely aligned tubules are connected by rows of highly periodic lateral arms about 13 nm long and 12 nm wide. The bundling activity of the enzyme seems to be specific since aldolase, which also interacts with microtubules, does not cross-link tubules, but it impedes the binding of the kinase to tubules. ATP, ADP and fructose bisphosphates inhibit the cross-bridges of microtubules by phosphofruktokinase to a different extent and concentration dependent manner. The kinase complexed with specific metabolites inducing distinct conformers does not interact with tubules. Microtubules cross-linked by the kinase became partly resistant to the depolymerizing action of vinblastine.

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.

Tubulin polymerization promoting protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays crucial role in the myelination of oligodendrocytes. Here we showed by CD, fluorescence, and NMR spectroscopies that Zn(2+) is the first ligand that induces considerable rearrangement of the disordered TPPP/p25. Zinc finger motif (His(2)Cys(2)) (His(61)-Cys(83)) was identified within the flexible region of TPPP/p25 straddled by extended unstructured N- and C-terminal regions. The specific binding of the Zn(2+) to TPPP/p25 induced the formation of molten globule but not that of a well-defined tertiary structure. The Zn(2+)-induced partially folded structure accommodating the zinc binding motif is localized at the single Trp(76)-containing region as demonstrated by fluorescence resonance energy transfer and quenching experiments. We showed that the Zn(2+)-induced change in the TPPP/p25 structure modified its interaction with tubulin and GTP coupled with functional consequences: the TPPP/p25-promoted tubulin polymerization was increased while the TPPP/p25-catalyzed GTPase activity was decreased as detected by turbidimetry and by malachite green phosphate release/(31)P NMR assays, respectively. The finding that the Zn(2+) of the bivalent cations can uniquely influence physiological relavant interactions significantly contributes to our understanding of the role of Zn(2+)-related TPPP/p25 processes in the differentiation/myelination of oligodendrocytes possessing a high-affinity Zn(2+) uptake mechanism.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease with variable extent of remyelination coupled with the differentiation of oligodendrocytes, in which Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) plays a crucial role. Previously we reported that the loss of TPPP/p25-positive oligodendrocytes in demyelinated lesions in the brain of MS patients could be a biomarker for MS [2]. In this work we tested the occurrence of TPPP/p25 in the cerebrospinal fluid (CSF) of MS patients, and by elaborating a sensitive assay for quantification of TPPP/p25 we showed that its level is significantly higher than in the case of non-MS patients. Patients with MS were diagnosed at the Department of Neurology, University of Szeged according to the clinical and laboratory diagnostic criteria of McDonald. In non-MS patients no significant pathological changes were detected on magnetic resonance imaging scans, while in MS patients multiple hyperintense T2 lesions in the white matter were detected. Kurtzke Expanded Disability Status Scale scores as well as IgG level and oligoclonal bands of MS patients were demonstrated. The sensitive assay elaborated in this study is based upon Western blot followed by chemiluminescent detection validated by human recombinant protein. The median TPPP/p25 contents in the CSF were 62.8 and 64.7 μg/L for patients with clinically isolated syndromes and relapsing remitting MS, respectively, while this value for non-MS patients was 27.9 μg/L. The enrichment of TPPP/p25 was independent of age, gender and the time period between lumbar puncture and relapse/shub. These data suggest that the TPPP/p25-based assay could be a powerful diagnostic test for MS patients.

TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of alpha-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of alpha-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 microM concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.

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.

A simple linearization procedure has been developed to determine the apparent dissociation constant of the interaction between antigen and antibody from the data of indirect, non-competitive enzyme-linked immunosorbent assays (ELISA). Applying this dissociation constant the binding constant of ligands to antigen can be determined and the quantitative evaluation of the competitive ELISA experiments makes it possible to analyse the affinity of antibody to antigen on the surface and in solution. The binding of the monospecific anti-calmodulin antibody to calmodulin and to solid-phase bound calmodulin has been tested by non-competitive and competitive assays. We have developed an experimental system where binding of the antibody to the solid-phase bound calmodulin has been studied under equilibrium conditions. Competitive ELISA experiments showed that the affinity of antibody to calmodulin on the surface and in solution was almost the same. The binding constant of a hypothalamic neuropeptide to calmodulin was determined using the quantitative ELISA approach. The neuropeptide was found to be of very high inhibitory potency (Kd = 2 nM) and competed with the antibody for calmodulin binding. This simple and sensitive procedure is suitable for screening molecules with anti-calmodulin activity and comparing their efficacy.

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.

There is increasing evidence that unfolded and misfolded proteins initiate a cascade of pathogenic protein-protein interactions that culminate in neuronal dysfunction. This is a multistep process which results in toxic protein aggregates; thus they are potent targets for development of early diagnosis and of drugs to improve therapies of conformational diseases. The hallmark proteins of these diseases such as Parkinson’s, Alzheimer’s or Huntington’s diseases, are α-synuclein, tau or mutant huntingtin, respectively, which do not have well-defined 3D structures and require protein partners to express their pathological functions. In this paper we review a new unstructured protein denoted Tubulin Polymerization Promoting Protein, TPPP/p25, from the discovery to its enrichment in human pathological inclusions characteristic for synucleinopathies with specific emphasis on its pursuits in single cells. There is a gappy area in the research of unfolded proteins referring to their structure-derived physiological and pathological functions. The studies of TPPP-homologous proteins at different levels of organization, molecular, cellular and tissue levels, rendered possible to reveal some TPPP/p25 specific structural and functional features, in addition to the general items for the role of the unfolded regions of the highly flexible proteins in their physiological and/or pathological functions.

New conformationally constrained adamantane 2-oxazoline building blocks 1-4 were synthesized and their antimicrotubule and anti trypanosomal potency was investigated. Although most of the new compounds affect tubulin polymerization, this does not make a major contribution to trypanocidal activity.

Tubulin polymerization promoting protein, (TPPP/p25), was identified as a brain-specific protein. The potential function of this protein resembled that of MAPs. It is mainly expressed in oligodendrocytes; however, immunopositivity was also detected in glial and neuronal inclusions in synucleinopathies. Here, we show that TPPP gene(s) are conserved in the genomes of ciliated organisms, but are lacking from the nonciliated ones. This recognition is based upon homologous gene sequence analysis, in silico comparative genomic studies, bioinformatic search and experimental evidence. Cilia (flagella) are microtubule-based cellular extensions of sensory and/or motile function. TPPP orthologs are among the only 16 genes that can be found in all ciliated organisms, suggesting that TPPP orthologs may be associated with a basic function of cilia.

Increasing evidence indicates that accumulation of unfolded/misfolded proteins results in protofibril and protein aggregate formation, inducing mitochondrial dysfunction and failure of synaptic, transport and other crucial physiological processes. Our knowledge on molecular defects of the energy metabolism in the pathogenesis of neurodegenerative diseases is scarce. One problem that investigators face is distinguishing primary from secondary events: is the impairment of energy production a consequence of the development of neurodegeneration, or it is an active contributor? Genomic and proteomic data provide useful information for understanding the structural and functional perturbations during the development of neurodegeneration, but the metabolomic alterations are much less disclosed. This paper reviews the direct and indirect role of the damage of energy metabolism, focusing on the metabolism of glucose as major energy source of the brain. Special attention is paid to the functional consequences of the associations of glycolytic enzymes with unfolded proteins that specifically determine the nature of the so-called “conformational diseases”. This review shows that the available data are largely inconclusive for the evaluation of the pathomechanism of energy production defects in human brains of patients of conformational diseases at system level, and the genuine need for development of rational models (biosimulation) of neuropathological conversion of glucose to ATP via glycolysis and oxidative phosphorylation.

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.

Summary Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.

Many glycolytic enzymopathies have been described that manifest clinically as chronic hemolytic anemia. One of these, triosephosphate isomerase (TPI) deficiency, is unique among the glycolytic enzyme defects since it is associated with progressive neurological dysfunction and frequently with childhood death. The physiological function of TPI is to adjust the rapid equilibrium between dihydroxyacetone phosphate and glyceraldehyde-3-phosphate produced by aldolase in glycolysis, which is interconnected to the pentose phosphate pathway and to lipid metabolism via triosephosphates. The TPI gene is well characterized; structure and function studies suggest that instability of the isomerase due to different mutations of the enzyme may underlie the observed reduced catalytic activity. Patients with various inherited mutations have been identified. The most abundant mutation is a Glu104Asp missense mutation that is found in homozygotes and compound heterozygotes. Two germ-line identical Hungarian compound heterozygote brothers with distinct phenotypes question the exclusive role of the inherited mutations in the etiology of neurodegeneration. This paper: (i) reviews our present understanding of TPI mutation-induced structural alterations and their pathological consequences, (ii) summarizes the consequences of TPI impairment in the Hungarian case at local and system levels, and (iii) raises critical questions regarding the exclusive role of TPI mutations in the development of this human disease. iubmb Life, 58: 703-715, 2006

Enolase is a glycolytic enzyme, expressed as cell-type specific isoforms in higher vertebrates. Herein we demonstrated for the first time that enolase isoforms interact with microtubules during muscle satellite cell differentiation. While in undifferentiated myoblasts the ubiquitous alphaalpha enolase isoform, expressed at high level, exhibited extensive co-localization with microtubules, the muscle-specific betabeta isoform, expressed at low level, did not. During differentiation, the level of beta subunit increased significantly; the alpha and beta enolase immunoreactivities were detected both in cytosol and along the microtubules. We identified tubulin from muscle extract as an interacting protein for immobilized betabeta enolase. ELISA and surface plasmon resonance measurements demonstrated the direct binding of enolase isoforms to tubulin with an apparent KD below the micromolar range, and indicated that the presence of 0.8 mM 2-phosphoglycerate abolished the interaction. Our data showed that, at various stages of myogenic differentiation, microtubules were decorated by different enolase isoforms, which was controlled by the abundance of both partners. We suggest that the binding of enolase to microtubules could contribute to the regulation of the dynamism of the cytoskeletal filaments known to occur during the transition from myoblast to myotubes.

There is evidence that ATP is an important extracellular factor regulating neutrophil functions. Indeed, extracellular ATP has been shown to stimulate neutrophil migration into sites of inflammation as well as adhesion of circulating cells to the endothelium1. This nucleotide also acts as a secretagogue for enhanced exocytosis of primary granules from neutrophils. Finally, it does improve the release of superoxide induced by fMLP, thereby acting as a priming agent for this response2. Extracellular ATP causes a transient increase in cytosolic level of Ca2+, which is known to be used as a second messenger to control many cellular processes including secretion, enzyme control, gene regulation, and apoptosis3. The Ca 2+ signal is a complex event that involves both intracellular Ca2+ pool release and extracellular Ca entry. Emptying of intracellular Ca2+ pools is the major trigger for activation of Ca entry during the generation of receptor-mediated Ca2+ signal. This capacitative mechanism, that has been termed store-operated Ca2+ entry, may be the basis by which the cells maintain elevated cytosolic free calcium concentration and replenish their intracellular Ca2+ stores in response to agonist stimulation. The importance of this signalling pathway has been recognised in numerous investigations and has received a great deal of attention.

Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his neurologically intact brother.

3'-(beta-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca(2+)-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation ((1)H-(15)N heteronuclear single quantum coherence) spectra of (15)N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.

International audience; The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transferred from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes--metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.