ABSTRACTS FROM RECENT PUBLICATIONS

Qilin Song, Deqiang Jing, and Charles K. Singleton. Tissue distribution of the thiamine transporter in normal and thiamine deficient rats (submitted)

Thiamine deficiency is characterized by differential vulnerabilities of tissue and cell types and inter-individual differences in susceptibility. Thiamine transport is an integral part of the tissue distribution of thiamine and its utilization, and it plays a role in thiamine deficiency. Herein, we have examined the tissue distribution of the thiamine transporter and the effects of thiamine deficiency on this distribution using pyrithiamine-induced thiamine deficiency in rats. The thiamine transporter was detected by immunoblots and immunohistochemistry. Relatively abundant levels were found in testes, ovaries, and liver and moderate amounts in spleen, kidney, and whole brain. Interestingly, little or no transporter was detected from skeletal muscle or heart tissue. Immunoblots indicated more or less equivalent amounts of transporter in all regions of the brain examined, except for somewhat low levels in the brain stem and high levels in cerebellum. Immunohistochemistry revealed subtle variations in levels within most brain regions. Thiamine deficiency resulted in a substantial increase in transporter levels within liver and more modest or only slight increases within other tissues. Both immunoblots and immunohistochemistry showed the level of the transporter was increased by thiamine deficiency in striatum, hippocampus and cerebral cortex, but no effect on the level of thiamine transporter was found in thalamus, hypothalamus, cerebellum and brainstem. Neither the distribution within the brain nor alterations of the distribution during thiamine deficiency correlated in any simple manner to the selective neurological damage brought about by thiamine deficiency.

Song, T. and Singleton, C.K. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate. BMC Biochemistry 2002, 3:8

Background
Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP also can be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant.
Results
Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 mM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values.
Conclusions
The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.

Singleton, C.K. and Martin, P. R. (2001). Molecular effects of thiamine deficiency. Current Molecular Medicine 1, 197-207

Thiamine is required for all tissues and is found in high concentrations in skeletal muscle, heart, liver, kidneys and brain. A state of severe depletion is seen in patients on a strict thiamine-deficient diet in 18 days, but the most common cause of thiamine deficiency is alcoholism. Thiamine diphosphate is the active form of thiamine, and it serves as a cofactor for several enzymes involved primarily in carbohydrate catabolism. The enzymes are important in the biosynthesis of a number of cell constituents, including neurotransmitters, and for the production of reducing equivalents used in oxidant stress defenses and in biosyntheses and for synthesis of pentoses used as nucleic acid precursors. Because of the latter fact, thiamine utilization is increased in tumor cells. Thiamine uptake by the small intestines and by cells within various organs is mediated by a saturable, high affinity transport system. Alcohol affects thiamine uptake and other aspects of thiamine utilization, and these effects may contribute to the prevalence of thiamine deficiency in alcoholics. The major manifestations of thiamine deficiency in humans involve the cardiovascular (wet beriberi) and nervous (dry beriberi, or neuropathy and/or Wernicke-Korsakoff syndrome) systems. A number of inborn errors of metabolism have been described in which clinical improvements can be documented following administration of pharmacological doses of thiamine, such as thiamine-responsive megaloblastic. Substantial efforts are being made to understand the genetic and biochemical determinants of inter individual differences in susceptibility to development of thiamine deficiency-related disorders and of the differential vulnerabilities of tissues and cell types to thiamine deficiency.

Transketolase. C. K. Singleton. Encyclopedia of Amolecular Medicine, in press.

Transketolase (EC 2.2.1.1) catalyzes the interconversion of sugars by transferring a 2 carbon ketol unit from a ketose donor substrate to an aldose acceptor substrate. The transfer is accomplished by the formation of a covalent intermediate between the ketol moiety and the thiazole ring of a thiamine diphosphate cofactor. The structure, cofactor binding sites, subunit interactions, and catalytic mechanism of transketolase have been studied extensively and are well understood. Transketolase catalyzes, in a rate limiting manner for the pathway, several reactions in the non-oxidative portion of the pentose phosphate pathway. By doing so transketolase, along with transaldolase, provides a reversible link between the oxidative portion of the pathway and glycolysis. The pentose phosphate pathway functions to form reducing equivalents and riboses for a number of cellular needs. Transketolase is a key enzyme in allowing flexibility of the pathway to meet varying needs of these two products in different cell types. Because of the role of transketolase in allowing rapidly dividing cells to produce large quantities of riboses for DNA synthesis, inhibition of transketolase is being examined as a potential anti-tumor treatment. During thiamine deficiency, transketolase activity, along with that of other thiamine diphosphate-utilizing enzymes, decreases in a cell-type dependent manner. The loss of activity of one or more of these enzymes contributes to the initiation of biochemical events that eventually lead to irreversible brain damage and dementia. A defect in a factor that modifies transketolase and perhaps other thiamine diphosphate-utilizing enzymes may result in an enhanced sensitivity to thiamine deficiency-induced neuropathy.

JNK1 is inactivated during thiamine deficiency-induced apoptosis in human neruoblastoma cells.
James Wang,, Hugh Fentress, Zhaolin Hua and Charles Singleton, J. Nutri. Biochem., 11, 208-215.

Thiamine deficiency results in selective neuronal damage. A number of mechanisms have been proposed to account for brain damage associated with thiamine deficiency and to account for the focal nature of the loss of neurons. One proposed mechanism is programmed cell death. We find efficient induction of apoptosis in human neuroblastoma cells when the cells were deprived of thiamine. Although extensive mitochondrial damage was seen, the release of cytochrome c was not the triggering mechanism for thiamine deficiency-induced apoptosis. Instead, the activity of the cJun amino terminal kinase Jnk1 was lost, and this loss correlated temporally with induction of apoptosis. The loss was specific for Jnk1 as Jnk2/3 activity remained unchanged. Loss of Jnk1 activity was not found in lymphoblasts, a cell type that did not undergo apoptosis when deprived of thiamine. The findings suggest that thiamine deficiency results in a cellular stress that brings about the loss of Jnk1 activity and the loss of its function of protecting cells from programmed cell death. We postulate that focal sensitivity to thiamine deficiency results, in part, from specific neuronal cell types being susceptible to the inactivation of Jnk1 in response to depletion of cellular thiamine.

Thiamine Deficiency Decreases in Steady-State mRNA Levels for Transketolase and Pyruvate Dehydrogenase but not for a-Ketoglutarate Dehydrogenase in Three Human Cell Types
J. Nutr. 128, 683-687 (1998)
Stevan R. Pekovich, Peter R. Martin, Charles K. Singleton

Reductions in the levels and activities of enzymes that utilize thiamine diphosphate (ThDP)2 as a cofactor are thought to be responsible for the tissue damage suffered during thiamine deficiency. Although loss of cofactor can account in part for loss of enzyme activity, thiamine and its phosphorylated derivatives may also regulate the expression of the genes encoding these proteins. To examine this possibility, steady-state mRNA levels were measured for three ThDP-dependent enzymes in human fibroblasts, lymphoblasts, and neuroblastoma cells cultured under thiamine sufficient and deficient conditions. In all three cell types, the mRNA levels of transketolase and the E1b subunit of pyruvate dehydrogenase complex were decreased during thiamine deficiency. In contrast, mRNA levels for a ThDP-binding subunit of a-ketoglutarate dehydrogenase, the E1 subunit, remained unchanged. These results indicate that, in humans, thiamine or a thiamine metabolite regulates the expression of some but not all genes encoding ThDP-utilizing enzymes.

Sensitivity to thiamine deficiency in cultured human cells is dependent on cell type and is enhanced in cells from thiamine-responsive megaloblastic anemia patients
J. Nutri. Biochem. 9, 215-222 (1998)
Stevan R. Pekovich, Vincenzo Poggi, Peter R. Martin, Charles K. Singleton

To address tissue-specific variation in sensitivity to thiamine deficiency, three human cell types were grown in medium with various thiamine concentrations. The activity of a cytosolic and a mitochondrial thiamine diphosphate-dependent enzyme was examined. Each cell type displayed a unique response to thiamine depletion with respect to a-ketoglutarate dehydrogenase and transketolase activity and to inhibition of cell growth. Loss of a-ketoglutarate dehydrogenase activity was similar in lymphoblasts and fibroblasts while loss of activity in neuroblastoma cells was significantly more resistant to thiamine depletion. Transketolase activity in neuroblastoma cells was only moderately resistant to thiamine depletion, with the activity in fibroblasts being the most and in lymphoblasts the least resistant. Total transketolase activity was 33% higher in fibroblasts than in lymphoblasts and neuroblastoma cells, indicating a differential requirement for production and maintenance of transketolase activity in this cell type. Compared to normal lymphoblasts, those derived from patients with thiamine-responsive megaloblastic anemia were 100 to 1000 times more sensitive to thiamine depletion. Although fibroblasts from these patients also demonstrated a 1000-fold increase in sensitivity with respect to transketolase activity, a-ketoglutarate dehydrogenase activity demonstrated no enhanced sensitivity. The results indicate a complex, cell-type dependent regulation of intracellular pools of thiamine and its phosphorylated derivatives in response to fluctuating extracellular thiamine levels.

ASPARTATE 155 OF HUMAN TRANSKETOLASE IS ESSENTIAL FOR THIAMINE DIPHOSPHATE-MAGNESIUM BINDING, AND COFACTOR BINDING IS REQUIRED FOR DIMER FORMATION
Biochem. et. Biophys. Acta 1341, 165-172 (1997)
James J.-L. Wang, Peter R. Martin, and Charles K. Singleton

Active human transketolase is a homodimeric enzyme possessing two active sites, each with a non-covalently bound thiamine diphosphate and magnesium. Both subunits contribute residues at each site which are involved in cofactor binding and in catalysis. His-tagged transketolase, produced in E. coli, was similar to transketolase purified from human tissues with respect to Km apps for cofactor and substrates and with respect to cofactor-dependent hysteresis. Mutation of aspartate 155, corresponding to a conserved aspartate residue among thiamine diphosphate-binding proteins, resulted in an inactive protein which could not bind the cofactor-magnesium complex and which could not dimerize. The results are consistent with the suggestion that aspartate 155 is an important coordination site for magnesium. In support of this interpretation, binding of cofactor by wild type apo-transketolase required the presence of magnesium. Additionally, monomeric apo-his-transketolase required both magnesium and cofactor binding for dimer formation.

Identification and characterization of the thiamine transporter gene of Saccharomyces cerevisiae
Gene 199, 111-121 (1997).
Charles K. Singleton

A positive selection scheme is described which selects for thiamine transporter clones. The scheme is based on the rescue of lethality, under nonpermissive conditions, of Saccharomyces cerevisiae strains that are conditional for thiamine biosynthesis and are defective in thiamine transport. Transport defective strains were generated by selection for resistance to the lethal thiamine analog, pyrithiamine. Pyrithiamine resistance was shown to be a recessive, single gene trait which resulted from the mutation of the thiamine transporter gene, as suggested by previous work. Conditional thiamine biosynthesis was generated by cloning THI4 , a thiamine biosynthetic gene, into a URA3 containing plasmid and transforming a strain disrupted in THI4. Thus, plating on 5-fluoroorotic acid causes the loss of thiamine synthesis ability. The gene for the yeast thiamine transporter, THI7, was cloned using this scheme. The predicted 598 amino acid transporter is a member of the major facilitator superfamily of transporters and thus possesses 12 transmembrane spanning segments with amino and carboxy termini intracellularly located. Several alterations in the coding region were characterized which result in greatly reduced thiamine transport ability. The level of transporter mRNA was found to be rapidly and dramatically reduced by the addition of thiamine to the growth medium.

A transketolase assembly defect in a Wernicke-Korsakoff syndrome patient.
Alcohol Clin Exp Res 21:576-580 (1997)
Wang JJ, Martin PR, Singleton CK

Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the
development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among
individuals are not understood. Investigations into these mechanisms have examined potential
variations in thiamine utilizing enzymes. Transketolase is a homodimeric enzyme containing two
molecules of noncovalently bound thiamine pyrophosphate. In the present study, we examined a
his-tagged human transketolase that was produced in and purified from Escherichia coli cells. Previous findings demonstrated that purified his-transketolase had a Km app for cofactor and a thiamine pyrophosphate-dependent lag period for attaining steady-state kinetics that was similar to transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells. Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced sensitivity to thiamine deficiency.

Conserved residues are functionally distinct within transketolases of different species.
Biochemistry 35:15865-15869 (1996)
Singleton CK, Wang JJ, Shan L, Martin PR

Most of the amino acid residues which interact with thiamine pyrophosphate are highly conserved
among enzymes which use this cofactor. The possible roles of several such residues in cofactor
binding, catalysis, and/or substrate binding were examined for human transketolase. Mutations in
H110 resulted in dramatic reductions to 2% or less of the normal activity. No alterations were found in the K(m)app's for the cofactor or for the donor and acceptor substrates. Alterations in Q428 resulted in a less severe loss of activity and also no changes in the K(m)app's. On the basis of the results, H110, an invariant residue, is proposed to function as a base which abstracts a proton from the protonated 4'-iminopyrimidine ring. The deprotonated 4'-imino moiety is required for generation of the C2-thiazolium carbanion which attacks the donor substrate. Interestingly, the function in the human enzyme of this invariant histidine is distinct from its role in yeast transketolase in which it aids in binding donor substrate and in subsequent catalytic events. Q428 is suggested to play a supportive role by stabilizing and orientating a water molecule which mediates the interaction between the 4'-amino group and H110. In other TPP-utilizing enzymes, the equivalent residue of Q428 is a histidine and is thought to deprotonate the 4'-amino group.