Further evidence for microtubule-independent dimerization of TPPP/p25.

Tubulin Polymerization Promoting Protein (TPPP/p25) is a brain-specific disordered protein that modulates the dynamics and stability of the microtubule network by its assembly promoting, cross-linking and acetylation enhancing activities. In normal brain it is expressed primarily in differentiated oligodendrocytes; however, at pathological conditions it is enriched in inclusions of both neurons and oligodendrocytes characteristic for Parkinson's disease and multiple system atrophy, respectively. The objective of this paper is to highlight a critical point of a recently published Skoufias's paper in which the crucial role of the microtubules in TPPP/p25 dimerization leading to microtubule bundling was suggested. However, our previous and present data provide evidence for the microtubule-independent dimerization of TPPP/p25 and its stabilization by disulphide bridges. In addition, our bimolecular fluorescence complementation experiments revealed the dimerization ability of both the full length and the terminal-free (CORE) TPPP/p25 forms, however, while TPPP/p25 aligned along the bundled microtubule network, the associated CORE segments distributed mostly homogeneously within the cytosol. Now, we identified a molecular model from the possible ones suggested in the Skoufias's paper that could be responsible for stabilization of the microtubule network in the course of the oligodendrocyte differentiation, consequently in the constitution of the myelin sheath.

Tubulin acetylation promoting potency and absorption efficacy of deacetylase inhibitors.

BACKGROUND AND PURPOSE: Histone deacetylase 6 (HDAC6) and silent information regulator 2 (SIRT2) control the dynamics of the microtubule network via their deacetylase activities. Tubulin polymerization promoting protein (TPPP/p25) enhances microtubule acetylation by its direct binding to HDAC6. Our objective was to characterize the multiple interactions of the deacetylases and to establish the inhibitory potency and the pharmacokinetic features of the deacetylase inhibitors, trichostatin A (TSA) and AGK2. EXPERIMENTAL APPROACH: The interactions of deacetylases with tubulin and TPPP/p25 were quantified by elisa using human recombinant proteins. The effect of inhibitors on the tubulin acetylation was established in HeLa cells transfected with pTPPP and CG-4 cells expressing TPPP/p25 endogenously by celisa (elisa on cells), Western blot and immunofluorescence microscopy. The pharmacokinetic features of the inhibitors were evaluated by in situ kinetic modelling of their intestinal transport in rats. KEY RESULTS: Deacetylases interact with both tubulin and TPPP/p25, notwithstanding piggy-back binding of HDAC6 or SIRT2 to the TPPP/p25-associated tubulin was established. Much higher inhibitory potency for TSA than for AGK2 was detected in both HeLa and CG-4 cells. Pioneer pharmacokinetic studies revealed passive diffusion and diffusion coupled with secretion for TSA and AGK2 respectively. Both inhibitors exhibited greater permeability than some other well-established drugs. CONCLUSIONS AND IMPLICATIONS: TPPP/p25-directed deacetylase inhibition provides mechanisms for the fine control of the dynamics and stability of the microtubule network. Deacetylase inhibitors with chemical structures similar to TSA and AGK2 appear to be excellent candidates for oral drug absorption.

Moonlighting microtubule-associated proteins: regulatory functions by day and pathological functions at night.

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.

Progress in the development of early diagnosis and a drug with unique pharmacology to improve cancer therapy.

Cancer continues to be one of the major health and socio-economic problems worldwide, despite considerable efforts to improve its early diagnosis and treatment. The identification of new constituents as biomarkers for early diagnosis of neoplastic cells and the discovery of new type of drugs with their mechanistic actions are crucial to improve cancer therapy. New drugs have entered the market, thanks to industrial and legislative efforts ensuring continuity of pharmaceutical development. New targets have been identified, but cancer therapy and the anti-cancer drug market still partly depend on anti-mitotic agents. The objective of this paper is to show the effects of KAR-2, a potent anti-mitotic compound, and TPPP/p25, a new unstructured protein, on the structural and functional characteristics of the microtubule system. Understanding the actions of these two potential effectors on the microtubule system could be the clue for early diagnosis and improvement of cancer therapy.

Interactions of enolase isoforms with tubulin and microtubules during myogenesis.

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.

New anti-mitotic drugs with distinct anti-calmodulin activity.

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: from unfolded protein to misfolding disease: prediction and experiments.

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 alpha-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 alpha-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.

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation.

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: kon, 2.4 x 10(4) M(-1) x s(-1); koff, 5.4 x 10(-3) s(-1); and Kd, 2.3 x 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.

Tubulin and microtubule are potential targets for brain hexokinase binding.

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 (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted 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.

Distinct behavior of mutant triosephosphate isomerase in hemolysate and in isolated form: molecular basis of enzyme deficiency.

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates. Enhanced attachment of mutant TPI to erythrocyte inside-out vesicles and to microtubules of brain cells was found when the binding was measured with TPIs in hemolysate. In contrast, there was no difference between the binding of the recombinant wild-type and Phe240Leu mutant enzymes. These findings suggest that the missense mutation by itself is not enough to explain the low catalytic activity and "stickiness" of mutant TPI observed in hemolysate. The activity of the mutant TPI is further reduced by its attachment to inside-out vesicles or microtubules. Comparative studies of the hemolysate from a British patient with Glu104Asp homozygosity and with the platelet lysates from the Hungarian family suggest that the microcompartmentation of TPI is not unique for the hemolysates from the Hungarian TPI-deficient brothers. The possible role of cellular components, other than the mutant enzymes, in the distinct behavior of TPI in isolated form versus in hemolysates from the compound heterozygotes and the simple heterozygote family members is discussed.

A new bis-indole, KARs, induces selective M arrest with specific spindle aberration in neuroblastoma cell line SH-SY5Y.

KARs, new semisynthetic antitumor bis-indole derivatives, were found to be inhibitors of tubulin polymerization with lower toxicity than vinblastine or vincristine, used in chemotherapy. Here, we compare the effect of KARs with those of vinblastine and vincristine on cell viability, cell proliferation, and cell cycle in neuroblastoma cell line (SH-SY5Y). At concentrations of the different compounds equivalent in causing 50% of inhibition of cell growth, KARs induced a complete arrest in the G2/M phase, whereas vinblastine and vincristine induced a partial arrest in both G0/G1 and G2/M. Moreover, a combination of KAR-2 and W13 (an anticalmodulin drug) qualitatively caused a similar arrest in both G0/G1 and G2/M than vinblastine. Levels of cyclin A and B1 were higher in KARs-treated cells than in vinblastine- or vincristine-treated cells. Cdc2 activity was much higher in KAR-2 than in vinblastine-treated cells, indicating a stronger mitotic arrest. The effect of KAR2 and vinblastine on microtubules network was analyzed by immunostaining with anti-tubulin antibody. Results indicated that KAR-2-induces the formation of aberrant mitotic spindles, with not apparent effect on interphase microtubules, whereas vinblastine partially destroyed interphase microtubules coexisting with normal and aberrant mitotic spindles.

Quantitative characterization of homo- and heteroassociations of muscle phosphofructokinase with aldolase.

Dissociation of purified phosphofructokinase accompanied with inactivation was analyzed in the absence and presence of aldolase and the data were compared with those obtained with muscle extract. The kinetics of the decrease in enzymatic activity was highly dependent on the dilution factor in both cases, but the inactivation appeared to be biphasic only with extract. The inactivation of the phosphofructokinase was impeded by addition of excess of aldolase. Time courses of kinase inactivation were fitted by alternative kinetic models to characterize the multiple equilibria of several homo- and hetero-oligomers of phosphofructokinase. The combination of modeling data obtained with purified and extract systems suggests that aldolase binds to an intermediate dimer of phosphofructokinase and within this heterocomplex the kinase is completely active. The intermediate dimer is stabilized by association with microtubules and the kinase activity decreased due to dilution can be recovered by addition of excess aldolase. In extract, the phosphofructokinase is of sigmoidal character (Hill coefficient of 2.3); the addition of excess exogenous aldolase to phosphofructokinase resulted in heterocomplex formation displaying Michaelian kinetics. The possible physiological relevance of heterocomplex formation of phosphofructokinase in muscle extract is discussed.

Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis.

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO4(2-) inhibit the tubulin polymerization at an IC50 of 14-24 microM with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 microM. BIAcore measurements for tubulin-tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO4(2-). No inhibitory effect of Cu2+ or CrO4(2-) on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO4(2-) ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10-14 microM with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

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.

Enhanced association of mutant triosephosphate isomerase to red cell membranes and to brain microtubules.

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 (<5%) TPI activity and 20- or 40-fold higher erythrocyte dihydroxyacetone phosphate levels as compared with normal controls. Our present studies with purified TPI and hemolysates revealed the binding of TPI, and the binding of human wild-type and mutant TPIs in hemolysate, to the red cell membrane, and the interference of binding with other hemolysate proteins. The binding of the mutant TPI is enhanced as compared with the wild-type enzyme. The increased binding is influenced by both the altered structure of the mutant and the changes in the red cell membrane. Compared with binding of glyceraldehyde-3-phosphate dehydrogenase, the isomerase binding is much less sensitive to ionic strength or blocking of the N-terminal tail of the band-3 transmembrane protein. The binding of TPIs to the membrane decreases the isomerase activity, resulting in extremely high dihydroxyacetone phosphate levels in deficient cells. In cell-free brain extract, tubulin copolymerizes with TPI and with other cytosolic proteins forming highly decorated microtubules as shown by immunoblot analysis with anti-TPI antibody and by electron microscopic images. The efficacy order of TPI binding to microtubules is propositus > 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.

Macromolecular compartmentation and channeling.

One of the accepted characterizations of the living state is that it is complex to an extraordinary degree. Since our current understanding of the living condition is minimal and fragmentary, it is not surprising that our first descriptions are simplistic. However, in certain areas of metabolism, especially those that have been amenable to experimentation for the longest period of time, the simplistic explanations have been the most difficult to revise. For example, current texts of general biochemistry still view metabolism as occurring by a series of independent enzymes dispersed in a uniform aqueous environment. This notion has been shown to be deeply flawed by both experimental and theoretical considerations. Thus, there is ample evidence that, in many metabolic pathways, specific interactions between sequential enzymes occur as static and/or dynamic complexes. In addition, reversible interactions of enzymes with structural proteins and membranes is a common occurrence. The interactions of enzymes give rise to a higher level of complexity that must be accounted for when one wishes to understand the regulation of metabolism. One of the phenomena that occurs because of sequential enzyme interactions is the process of channeling. This article discusses enzyme interactions and channeling and summarizes experimental and theoretical results from a few well-studied examples.

Substrate channeling.

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.

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.