Jan Czerniecki

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells.

Background Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. Methodology and Principal Findings Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. Conclusions and Significance The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

AbstractIn most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.

Neuronal localization of the 25-kDa specific thiamine triphosphatase in rodent brain.

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate donor for the phosphorylation of certain proteins; this may be part of a new signal transduction pathway. We have recently characterized a highly specific 25-kDa thiamine triphosphatase (ThTPase) that is expressed in most mammalian tissues. The role of this enzyme may be the control of intracellular concentrations of ThTP. As the latter has been considered to be a neuroactive form of thiamine, we have studied the distribution of ThTPase mRNA and protein in rodent brain using in situ hybridization and immunohistochemistry. With both methods, we found the strongest staining in hippocampal pyramidal neurons, as well as cerebellar granule cells and Purkinje cells. Some interneurons were also labeled and many ThTPase mRNA-positive and immunoreactive cells were distributed throughout cerebral cortical gray matter and the thalamus. White matter was not significantly labeled. ThTPase immunoreactivity seems to be located mainly in the cytoplasm of neuronal perikarya. Immunocytochemical data using dissociated cultured cells from hippocampal and cerebellum showed that the staining was more intense in neurons than in astrocytes. The protein was rather uniformly located in the perikarya and dendrites, suggesting that ThTP and ThTPase may play a general role in neuronal metabolism rather than a specific role in excitability. There was no apparent correlation between ThTPase expression and selective vulnerability of certain brain regions to thiamine deficiency.

Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase

BACKGROUND Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. METHODS Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. RESULTS Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. CONCLUSIONS The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. GENERAL SIGNIFICANCE We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.

Kinetic and spectral investigation of allosteric interaction of coenzymes with 2-oxo acid dehydrogenase complexes

The possible role of thiamine pyrophosphate (TPP) in the regulation of both multienzyme pyruvate dehydrogenase complex (PDC) and 2-oxoglutarate dehydrogenase complex (OGDC) has been investigated by kinetic and spectral methods. The purified PDC and OGDC from animal heart muscle were near saturated with endogenous TPP. The PDC containing the bound coenzyme showed hysteretic behaviour manifested in a lag phase of the catalysed reaction after the contact of PDC with substrates. Exogenous TPP added to the full reaction medium led to a disappearance of the lag phase and to strong reduction of the Michaelis constant (Km) value for pyruvate, and more moderate decrease of Km for both coenzyme A and NAD. In the case of OGDC exogenous TPP also decreased S0.5 (Km) for substrate 2-oxoglutarate. In addition, exogenous TPP changed both the UV and circular dichroism spectra of PDC and last one of OGDC, and lowered the fluorescence emission of the multienzyme complexes containing bound molecules of endogenous coenzyme in their active sites. Thiamine pyrophosphate seems to play, besides its coenzyme function, the role of positive allosteric effector which causes conformational changes of the multienzyme complexes and increases their affinity to substrates. q 2002 Elsevier Science B.V. All rights reserved.

Changes in ECG and enzyme activity in rat heart after myocardial infarction : effect of TPP and MnCl2

Heart infarction is one of the main causes of death in the human population. Assurance of a sufficient level of bioenergetic processes is very important for the heart after infarction. Mn2+ as well as thiamine pyrophosphate (TPP) are positive effectors of the pyruvate dehydrogenase complex (PDH) and the 2-oxoglutarate dehydrogenase complex (OGDH), both of which play a very important role in the Krebs cycle. Thus, we have established the effect of MnCl2 (10mg/kg) and TPP (20mg/kg)-4 injections every 12 h-on the activity of PDH, OGDH, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). Additionally, we perform an analysis of ECG to affirm the changes in the heart electrophysiology of healthy rats after MnCl2 and TPP treatment. We then analyzed changes in the activity of these enzymes after experimental myocardial infarction in rats. We observed a decrease of OGDH and MDH activity in rat hearts after infarction in comparison, with sham-operated rats. Treatment of healthy rats with MnCl2 caused an increase of OGDH activity. Moreover both MnCl2 and TPP caused an increase of PDH activity and a decrease of MDH activity (TPP revealed a stronger effect). We found no changes in LDH activity. Electrocardiography data showed a slight shortening of the QT interval and an enhanced heartbeat rate after treatment with MnCl2. TPP caused only elongation of the QT interval. In conclusion, application of MnCl2 enhanced the activity of some very important enzymes in the respiration process (PDH and OGDH). This effect, connected with enhanced heartbeat and a slightly shortened ventricle relaxation, may have potential application during the key period of convalescence following heart infarction.ResumenTras infarto de miocardio, es de gran importancia el mantenimiento de un adecuado nivel energético. El catión Mn2+ y la tiamina pirofosfato (TPP) afectan positivamente la actividad de los complejos piruvato deshidrogenasa (PDH) y 2-oxoglutarato deshidrogenasa (OGDH), con importante papel en el ciclo de Krebs. Asi, se ha estudiado el efecto del MnCl2 (10 mg/kg) y la TPP (20 mg/kg), 4 inyecciones cada 12 h, sobre la actividad PDH, OGDH, lactato deshidrogenasa (LDH) y malato deshidrogenasa (MDH), tanto en ratas sanas como tras infarto de miocardio. Además se ha analizado el ECG de ratas sanas tras tratamiento con MnCl2 y TPP. Los resultados muestran descenso en la actividad OGDH y MDH en ratas que han sufrido infarto respecto de sus controles. En ratas sanas, el MnCl2 incrementa la actividad OGDH y, tanto MnCl2 como TPP, incrementan la actividad PDH, Los datos electrocardiográficos muestran un ligero acortamiento del intervalo QT, con incremento de la frecuencia cardiaca tras el tratamiento con MnCl2. La TPP causa sólo alargamiento del intervalo QT. Los resultados sugieren una posible aplicación potencial del MnCl2 durante el período de convalecencia posterior al infarto de miocardio.