Localization of Thiamine-Binding Protein in Synaptosomes from the Rat Brain

Using preparations of synaptosomes and subsynaptosomal fractions from the rat brain, we studied the localization of thiamine-binding protein (TBP) in the subcellular structures of the neurons. In addition, we studied the distribution in synaptosomes of two types of activity typical of TBP (thiamine triphosphatase and thiamine-binding activities), as well as the effects of factors destroying the plasma membrane of synaptosomes on binding of [¹⁴C]thiamine with the latter. We found that the thiamine-associated activity of TBP was the highest in fractions of the synaptic vesicles and plasma membranes. Hydrolysis of thiamine triphosphate was also most active in these structures. Our results allow us to conclude that TBP is localized mostly in the synaptic vesicles and plasma membranes of synaptosomes.     

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.     

Optical Characteristics of Thiamine in Model Systems and in Holoenzyme

The optical properties of thiamine diphosphate-dependent enzymes change significantly on their interaction with cofactors (thiamine, bivalent metal ions) and substrates. These changes are connected with structural alterations of the active site and the mechanism of its functioning, and in some cases they reflect changes in the optical properties of the coenzyme itself within the protein. The use of optical characteristics, especially together with model systems, appeared to be a rather promising approach for investigation of the active site of thiamine diphosphate-dependent enzymes and the mechanism of its functioning. So, it seemed to be useful to summarize the literature data concerning the optical characteristics of thiamine (thiamine diphosphate) in model systems and the efficiency of their application for study of thiamine diphosphate-dependent enzymes.     

Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes

Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family.     

Quantitation of plasma thiamine,related metabolites and plasma protein oxidative damage markers in children with autism spectrum disorder and healthy controls

Abstract

Aims/hypothesis: To assess thiamine and related metabolite status by analysis of plasma and urine in autistic children and healthy controls, correlations to clinical characteristics and link to plasma protein markers of oxidative damage.

Methods: 27 children with autism (21 males and 6 females) and 21 (15 males and 6 females) age-matched healthy control children were recruited. The concentration of thiamine and related phosphorylated metabolites in plasma and urine and plasma protein content of dityrosine, N-formylkynurenine and 3-nitrotyrosine was determined.

Results: Plasma thiamine and thiamine monophosphate concentrations were similar in both study groups (median [lower–upper quartile]): autistic children – 6.60?nM (4.48–8.91) and 7.00?nM (5.51–8.55), and healthy controls – 6.82?nM (4.47–7.02) and 6.82?nM (5.84–8.91), respectively. Thiamine pyrophosphate (TPP) was decreased 24% in autistic children compared to healthy controls: 6.82?nM (5.81–8.52) versus 9.00?nM (8.41–10.71), p?<?.01. Urinary excretion of thiamine and fractional renal clearance of thiamine did not change between the groups. No correlation was observed between clinical markers and the plasma and urine thiamine concentration. Plasma protein dityrosine content was increased 88% in ASD. Other oxidative markers were unchanged.

Conclusions/interpretation: Autistic children had normal plasma and urinary thiamine levels whereas plasma TPP concentration was decreased. The latter may be linked to abnormal tissue handling and/or absorption from gut microbiota of TPP which warrants further investigation. Increased plasma protein dityrosine may reflect increased dual oxidase activity in response to change in mucosal immunity and host–microbe homeostasis.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Non-coenzyme role of vitamin B1 in RANKL-induced osteoclastogenesis and ovariectomy induced osteoporosis

Vitamins B are co-enzymes participating in energy metabolic pathways. While some vitamins B are known affecting bone homeostasis, the effects of vitamin B1 (thiamine) on bone health remains unclear. In our study, we used cell counting kit-8, tartrate-resistant acid phosphatase stain, actin cytoskeleton stain, and pit formation assay to evaluate the effect of thiamine on osteoclast differentiation, formation, and function, respectively. Then we used dichloro-dihydro-fluorescein diacetate assay to investigate reactive oxygen species (ROS) generation and removal. Osteoporosis model by ovariectomy was established for animal experiments. We found that thiamine had inhibitory effect on osteoclast differentiation. And its inhibitory role on osteoclast differentiation is in a dose-dependent way. Mechanistically, ThDP suppresses intracellular ROS accumulation and unfolded protein response signaling during osteoclastogenesis via inhibiting Rac-Nox1/2/4 and intracellular inositol-requiring protein-1α/X-box-binding protein pathways, respectively. Osteoporotic mice treated with thiamine rich dietary showed better bone strength relative to thiamine deficient dietary. Our study explored the non-coenzyme inhibitory functions of B1 vitamin in receptor activator of nuclear factor κB ligand induced osteoclastogenesis and uncovered the significance of B1 vitamin in bone health.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine deficiency encephalopathy: 3. Transketolase

Chronic thiamine deprivation in the rat leads to ataxia, loss of righting reflex and neuropathological damage to lateral vestibular nucleus. Before onset of neurological symptoms, transketolase (TK) activities were found to be selectively reduced by 25% in lateral vestibular nucleus and surrounding pons. Further progression of thiamine deprivation resulted in a generalized reduction in TK activity. Measurement of enzyme activity in the presence of added TPP cofactor in vitro did not lead to normalisation of enzyme activities suggesting loss of apoenzyme. Administration of thiamine to symptomatic thiamine-deprived rats resulted in reversal of neurological symptoms and to normalisation of defective TK activities in less vulnerable structures such as cerebral cortex striatum and hippocampus; reduction of TK activity, however, persisted in brainstem and cerebellar regions. Pyrithiamine treatment results, within 3 weeks, in loss of righting reflex, convulsions and more widespread neuropathological damage compared to that observed following thiamine deprivation. TK activity was found to be significantly decreased before the onset of neurological symptoms in all brain regions and appearance of symptoms was accompanied by more severe reductions of TK. In contrast to chronic thiamine deprivation, TK activities following pyrithiamine treatment were: (i) equally reduced in magnitude in vulnerable and non-vulnerable brain structures, (ii) unchanged following reversal of neurological abnormalities by thiamine administration.     

Synthesis of thiamine triphosphate in rat brain in vivo

Thiamine metabolism in vivo was studied by intracerebroventricular injection of labeled thiamine in rat brain. Labeled thiamine was found to be rapidly converted to the phosphorylated thiamine esters. The distribution of the radioactive thiamine compounds was reached to steady state at 3 hr after injection: thiamine, thiamine monophosphate, thiamine pyrophosphate, and thiamine triphosphate were 8–12%, 12–14%, 72–74%, and 2–3%, respectively, in cerebral cortex. The presence of labeled thiamine triphosphate in the brain was further confirmed by the treatment with thiamine triphosphatase which had an absolute substrate specificity for thiamine triphosphate. These results suggest that thiamine triphosphate is synthesized in vivo in rat brain.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

Tissue difference in cellular localization of thiamine phosphate esters

1. Thiamine phosphate levels were determined in the soluble and particulate fractions of various rat tissues. 2. There was marked tissue difference in the cellular localization of thiamine phosphates. 3. Brain thiamine triphosphate was localized only in the particulate fraction, whereas skeletal muscle thiamine triphosphate was in the soluble fraction as a protein-unbound form.     

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.     

Thiamine limitation determines the transition from aerobic to fermentative-like metabolism in Rhizobium etli CE3

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169∷ miniTn 5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-β-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.     

Temporal changes in Sander vitreus egg thiamine levels

Thiamine pyrophosphate was the predominant form of thiamine present initially in walleye Sander vitreus eggs from two spawning locations in Lake Oahe, South Dakota, U.S.A. Total thiamine content in the eggs at fertilization was 5·18 and 7·97 nmol g⁻¹ for eggs from the Moreau and Grand River spawning sites respectively, and egg thiamine content in all its forms dropped dramatically at the next sampling period of 48 temperature units (TU). Thiamine values did not significantly drop after the 48 TU period, but mean total thiamine composition was < 0·9 nmol g⁻¹ at the last sampling date (156 TU) just prior to hatching.     

A novel thiamine-derived pigment,pyrizepine, formed by the Maillard reaction

To find a Maillard pigment derived from thiamine, a solution containing glucose and thiamine was heated and analyzed with high-performance liquid chromatography equipped with diode-array detection. As a result, a unique peak showing an absorption maximum at 380 nm was detected. This peak was then isolated from a reaction solution containing glucose, lysine and thiamine, and was identified as 1-(2-methyl-6,9-dihydro-5H-pyrimido[4,5-e][1,4]diazepin-7-yl)ethan-1-one using instrumental analyses. This compound, named pyrizepine, was a novel yellow pigment having a fused ring consisting of pyrimidine and diazepine. Pyrizepine was a major low-molecular-weight pigment in the reaction solution. The structure suggests that pyrizepine is formed by condensation reaction between a degradation product of thiamine and a tetrosone derivative formed from glucose by the Maillard reaction.     

Thiamine Triphosphate and Membrane-Associated Thiamine Phosphatases in the Electric Organ of Electrophorus electricus

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.     

Thiamine phosphates and regulation of the pyruvate dehydrogenase complex activity in rat liver mitochondria

The possibility of thiamine phosphates to participate in the regulation of pyruvate dehydrogenase complex activity on the level of isolated mitochondria is studied. It is shown that an increase in the thiamine diphosphate concentration in incubation medium produces no significant changes in the pyruvate dehydrogenase activity of mitochondria. The pyruvate dehydrogenase activity decreases when mitochondria are incubated with thiamine triphosphate or ATP under different conditions. Thiamine triphosphate is not able to replace ATP in kinase reaction of the isolated complex, but it inhibits reactivation of the complex with exogenase phosphatase; under the same conditions thiamine diphosphate activates phosphatase. Analysis of these data leads to conclusion that under native conditions an increase of the intramitochondrial thiamine triphosphate concentration can produce a drop in the pyruvate dehydrogenase complex activity by inhibition of the phosphatase reaction.     

The effects of endurance training and thiamine supplementation on anti-fatigue during exercise

The purpose of this study was to find the effect of endurance training and thiamine supplementation on anti-fatigue during the exercise. Each nine students from K Women’s University went through three cross-over treatments: placebo treatment, training treatment and thiamine treatment. Training treatment was performed with bicycle ergometer exercise for four weeks (five days per week). Each exercise was performed for an hour with intensity set at 70% (50rpm) of maximal oxygen uptake. Thiamine treatment group was given 10mg of thiamine tetrahydrofurfuryl disulfide per one kilogram for four weeks. The bicycle ergometer exercise was performed at 70% of maximal oxygen uptake in exercise intensity which 60 minutes of exercise was performed at 50rpm . Lactate concentration was significantly decreased during 15 to 30 minutes of exercise for those with training treatment and 15 to 60 minutes of exercise for those with thiamine treatment compared to placebo treatment group. Ammonia concentration was significantly decreased during 15 to 60 minutes of exercise and 15 to 30 minutes of recovery for those with training and thiamine treatment compared to placebo treatment. Resting blood thiamine concentrations of placebo treatment were significantly lower than training treatment. 60 minutes after the exercise, plasma thiamine concentration was significantly increased in all treatment group. To sum up the previous, thiamine intake during exercise positively benefits carbohydrate metabolism in a way that will decrease lactate concentration, ammonia concentration, and anti- fatigue by reducing the RPE. Therefore, we can consider thiamine intake to be utilized as similar benefits as endurance training.     

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.