Combined Enhancement of Microtubule Assembly and Glucose Metabolism in Neuronal Systems in Vitro: Decreased Sensitivity to Copper Toxicity

Liliom, Károly; Wágner, Gábor; Kovács, János; Comin, Begoña; Cascante, Marta; Orosz, Ferenc; Ovádi, Judit

doi:10.1006/bbrc.1999.1547

Cited by 25 publications

(33 citation statements)

References 23 publications

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“…The general properties of the temperature-induced self-assembly of HsCen2, observed in these experiments, are similar to those of the polymerization process observed for other biomolecules, such as hemoglobin S (18), actin (19 -21), or tubulin (22)(23)(24).…”

Section: Resultssupporting

confidence: 78%

Calcium-dependent Self-assembly of Human Centrin 2

Tourbez

Firanescu²,

Yang

et al. 2004

Journal of Biological Chemistry

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Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca 2؉ -sensitive fibers and their precise role in centrosome duplication are not known. To explore the possible structural role of HsCen2, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. Using light scattering experiments, we investigated the temporal evolution of the assembly process and characterized the dependence on various chemical and physical factors, including temperature, di-cation concentration, ionic strength, protein concentration, and pH. The reversible self-assembly revealed many features of a large-size protein polymerization, with nucleation and elongation steps. Kinetic and equilibrium experiments show that a hydrophobic fluorescent probe (ANS) inhibits the polymerization by interfering with the nucleation step, probably through interactions with the apolar exposed sites on the protein surface. A truncated form of HsCen2, lacking the first 25 residues (⌬25HsCen2), shows no detectable self-assembly, pointing to the critical role played by the N-terminal fragment in the supermolecular organization of HsCen2. As revealed by isothermal titration experiments, the isolated N-terminal domains bind with a significant affinity (2 ؋ 10 5 M ؊1 ) to preformed oligomers of ⌬25HsCen2 through an entropy-driven mechanism.

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Section: Resultssupporting

confidence: 78%

Calcium-dependent Self-assembly of Human Centrin 2

Tourbez

Firanescu²,

Yang

et al. 2004

Journal of Biological Chemistry

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“…We have previously demonstrated that brain and neuroblastoma but not muscle extract stimulate paclitaxel-assisted tubulin polymerization as followed by turbidimetry (19). The partially purified but especially the isolated TPPP͞p25 protein initiates the assembly of microtubule-associated protein-free tubulin even in the absence of paclitaxel.…”

Section: Resultsmentioning

confidence: 97%

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation

Tirián

Hlavanda

Oláh

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

116

136

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Recently, we isolated from bovine brain a protein, TPPP͞p25 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 TPPP͞p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 ؋ 10 4 M ؊1 ⅐s ؊1 ; koff, 5.4 ؋ 10 ؊3 s ؊1 ; and Kd, 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 TPPP͞p25 into cleavage Drosophila embryos expressing tubulin-GFP fusion protein reveals that TPPP͞p25 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 TPPP͞p25 both in vitro and in vivo.T he cytoplasm of eukaryotic cells contains an elaborate network of cytoskeletal elements, consisting of actin and intermediate filaments and microtubules (MTs) engaged in a variety of cell functions, such as the extension and guidance of neurons or the formation of mitotic spindles required for chromosomal segregation. The polymerization dynamics of tubulin to MTs is under strict control (1). Numerous proteins have been reported to interact with the MTs as positive regulators of MT assembly (microtubule-associated proteins), either by promoting the polymerization of tubulin or by stabilizing MTs (1, 2). Only a few proteins are known to act as destabilizers, such as Op18͞stathmin, katanin, and some kinesin-like proteins (1, 2). We have reported recently that the M1 isoform of pyruvate kinase and Dictyostelium discoideum phosphofructokinase inhibit tubulin polymerization or promote disassembly of the MTs to thread-like oligomers in vitro (3-5).Recently, from bovine brain, we isolated and identified a protein that we denoted TPPP͞p25 (6), which corresponded to p25 (NCBI accession no. 2498194). This protein is a heat stable, cationic protein that induces polymerization of tubulin into unusual forms or bundling of paclitaxel-stabilized MT. The TPPP͞p25 protein was partially copurified with a tau kinase (7). The bovine TPPP͞p25-coding gene has been cloned (8), and recently the human homologue of TPPP͞p25 was cloned and mapped to the p15.3 region of chromosome 5. The identity between the bovine and human TPPP͞p25 proteins is 90% (9). This protein was also described as p24, a heat-resistant glycogen synthase kinase-3 inhibitor (10). It is important to note that TPPP͞p25 differs completely from the extensively characterized protein p25, which is a truncated form of p35 that deregulates cyclin-dependent kinase-5 activity by causing prolonged activation and m...

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“…28 Similar stimulating effects on tubulin polymerization were detected by the addition of normal brain or neuroblastoma cell extracts but not with muscle cell extracts. 29 Differences in the enhancement of tubulin polymerization by wild-type and mutant enzymes are documented in Figure 1B. The mutant enzyme is more efficient in augmenting the MT assembly.…”

Section: Consequences Of the Heteroassociationsmentioning

confidence: 95%

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

Orosz

Oláh

Alvarez

et al. 2001

Blood

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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.

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Combined Enhancement of Microtubule Assembly and Glucose Metabolism in Neuronal Systems in Vitro: Decreased Sensitivity to Copper Toxicity

Cited by 25 publications

References 23 publications

Calcium-dependent Self-assembly of Human Centrin 2

Calcium-dependent Self-assembly of Human Centrin 2

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation

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

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