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Ferenc Orosz

Publications and source records attributed to Ferenc Orosz.

10 recordsLinked to original sources
PubMed2006

Tubulin polymerization promoting proteins (TPPPs): members of a new family with distinct structures and functions.

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

Amino Acid Sequence↗
PubMed2006

Interaction of TPPP/p25 protein with glyceraldehyde-3-phosphate dehydrogenase and their co-localization in Lewy bodies.

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

Aged↗
PubMed2005

Triosephosphate isomerase deficiency: consequences of an inherited mutation at mRNA, protein and metabolic levels.

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.

Alleles↗
PubMed2004

Methotrexate inhibits the glyoxalase system in vivo in children with acute lymphoid leukaemia.

The inhibition of glyoxalase I leads to antitumour activity through the accumulation of methylglyoxal. Our earlier observations suggested that methotrexate (MTX) may affect the glyoxalase system. This prompted a serial study of the drug on this metabolic pathway. Ten children with acute lymphoid leukaemia (ALL), admitted to our department between January 2002 and July 2003, were enrolled. Plasma D-lactate was assayed before, 24 and 72 h after the start of four consecutive MTX infusions (5 g/m(2)/24 h) in each patient. Inhibition of glyoxalase I was tested in vitro, using human erythrocyte lysates and yeast enzyme. The elevated initial plasma D-lactate levels (P<0.02) fell significantly (P<0.001) in response to 24 h MTX infusions. In vitro, MTX, folic and folinic acids inhibited the activity of glyoxalase I. Thus, MTX seems to affect the alpha-oxoaldehyde metabolism in vivo, as a likely consequence of glyoxalase I inhibition. This action probably contributes to the anticancer activity and toxicity of the drug.

Antimetabolites, Antineoplastic↗
PubMed2003

Glucose conversion by multiple pathways in brain extract: theoretical and experimental analysis.

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.

Animals↗
PubMed2002

A simple method for the determination of dissociation constants by displacement ELISA.

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.

Antibodies↗
PubMed2002

Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations.

Previously, we have demonstrated the presence of a protein factor [tubulin polymerization perturbing protein (TPPP)] in brain and neuroblastoma cell but not in muscle extract that uniquely influences the microtubule assembly. Here we describe a procedure for isolation of this protein from the cytosolic fraction of bovine brain and present evidence that this protein is a target of both tubulin and microtubules in vitro. The crucial step of the purification is the cationic exchange chromatography; the bound TPPP is eluted at high salt concentrations, indicating the basic character of the protein. By IDA-nanoLC-MS analysis of the peptides extracted from the gel-digested purified TPPP, we show the presence of a single protein in the purified fraction that corresponds to p25, a brain-specific protein the function of which has not been identified. Circular dichroism data have revealed that, on one hand, the alpha-helix content of p25 is very low (4%) with respect to the predicted values (30-43%), and its binding to tubulin induces remarkable alteration in the secondary structure of the protein(s). As shown by turbidimetry, pelleting experiments, and electron microscopy, p25 binds to paclitaxel-stabilized microtubules and bundles them. p25 induces formation of unusual (mainly double-walled) microtubules from tubulin in the absence of paclitaxel. The amount of aberrant tubules formed depends on the p25 concentration, and the process occurs at substoichiometric concentrations. Our in vitro data suggest that p25 could act as a unique MAP in vivo.

Animals↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMedDate not supplied

Functional aspects of cellular microcompartmentation in the development of neurodegeneration: mutation induced aberrant protein-protein associations.

Research in the last 10 years has revealed that the development of neurodegeneration is a multistep process during which one or few specific mutant protein species of altered conformation initiate aberrant protein-protein interactions resulting in aggregates forming plaques. This review focuses on the heteroassociations of the mutant proteins with subcellular structures, such as cytoskeleton, cell membranes or with glycolytic enzymes, which may be crucial in the initiation of neurodegeneration such as in Huntington's disease or Alzheimer's disease. Triosephosphate isomerase enzymopathy is a unique glycolytic enzyme deficiency coupled with neurodegeneration. We present data on the mutation induced misfolding process, which likely plays a crucial role in the enhanced associations of the enzyme with the truncated fragment of the isomerase, with the red cell membrane or with the microtubular network. On the basis of our recent clinical and experimental results obtained with two compound heterozygote Hungarian brothers it became obvious that the mutations alone are not sufficient to explain the development of the neurological sympthomes. This underscores the fact that the mutations alone are not enough for the development of the clinical phenotype of a disease.

Animals↗