Molecular mechanisms of thiamine utilization.

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 in affluent countries 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 anemia. 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.

JNK1 is inactivated during thiamine deficiency-induced apoptosis in human neuroblastoma cells.

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 found 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; 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. These 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.

The histidine kinase dhkC regulates the choice between migrating slugs and terminal differentiation in Dictyostelium discoideum.

An early decision that a newly formed aggregate of Dictyostelium cells must make is whether to form a migrating slug or to proceed through culmination, the process of forming the mature fruiting body. The choice between these alternative morphological pathways is influenced by external and internal cues. dhkC was identified as a potential hybrid sensor kinase possessing domains homologous to the histidine kinase and receiver motifs of two-component signaling systems. Null strains of dhkC show a rapidly developing phenotype for aggregation through finger formation, and culmination commences immediately thereafter and proceeds at a normal rate to generate typical fruiting bodies. Ammonia, an endogenous regulator of the slug versus culmination choice, results in a prolonged slug stage for wild-type strains while the dhkC- strain bypasses the slug stage in the presence or absence of ammonia. Conversely, expression in wild-type cells of a modified DHKC protein composed of only the histidine kinase domain results in normal timing through early aggregation, but subsequent development is significantly delayed. The resulting fingers, once formed, readily convert to slugs that do not undergo culmination but instead migrate until their energy sources are depleted. The slugger phenotype is dependent on the presence of a functional response regulator REGA, and it is rescued by exogenously supplied cAMP. Together, the results indicate that DHKC contributes to the integration of environmental and cellular signals so that the appropriate choice is made between slug formation and culmination. We suggest that DHKC may function as a sensor for ammonia, and that it is the initial component of a phosphorelay signaling system that may modulate the activity of cAMP-dependent protein kinase to either inhibit or promote culmination. Additionally, dhkC- spores were found to be defective in germination, indicating a role for the DHKC signaling pathway in activating spore germination.

The hybrid histidine kinase dhkB regulates spore germination in Dictyostelium discoideum.

Spore germination is a defined developmental process that marks a critical point in the life cycle of Dictyostelium discoideum. Upon germination the environmental conditions must be conducive to cell growth to ensure survival of emerged amoebae. However, the signal transduction pathways controlling the various aspects of spore germination in large part remain to be elucidated. We have used degenerate PCR to identify dhkB, a two-component histidine kinase, from D. discoideum. DhkB is predicted to be a transmembrane hybrid sensor kinase. The dhkB-null cells develop with normal timing to give what seem to be mature fruiting bodies by 22 to 24 h. However, over the next several hours, the ellipsoidal and encapsulated spores proceed to swell and germinate in situ within the sorus and thus do not respond to the normal inhibitors of germination present within the sorus. The emerged amoebae dehydrate due to the high osmolarity within the sorus, and by 72 h 4% or less of the amoebae remain as spores, while most cells are now nonviable. Precocious germination is suppressed by ectopic activation of or expression of cAMP-dependent protein kinase A. Additionally, at 24 h the intracellular concentration of cAMP of dhkB- spores is 40% that of dhkB+ spores. The results indicate that DHKB regulates spore germination, and a functional DHKB sensor kinase is required for the maintenance of spore dormancy. DHKB probably acts by maintaining an active PKA that in turn is inhibitory to germination.

Thiamine deficiency decreases steady-state transketolase and pyruvate dehydrogenase but not alpha-ketoglutarate dehydrogenase mRNA levels in three human cell types.

Reductions in the levels and activities of enzymes that utilize thiamine diphosphate (ThDP) 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 for three ThDP-dependent enzymes were measured in human fibroblasts, lymphoblasts and neuroblastoma cells cultured under conditions of thiamine sufficiency and deficiency. In all three cell types, the mRNA levels of transketolase and the E1beta subunit of pyruvate dehydrogenase complex were lower in thiamine-deficient cultures. In contrast, mRNA levels for a ThDP-binding subunit of alpha-ketoglutarate dehydrogenase, the E1 subunit did not differ. These results indicate that thiamine or a thiamine metabolite regulates the expression in humans of some, but not all, genes encoding ThDP-utilizing enzymes.

Aspartate 155 of human transketolase is essential for thiamine diphosphate-magnesium binding, and cofactor binding is required for dimer formation.

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.

A positive selection scheme is described that selects for thiamine transporter clones. The scheme is based on the rescue of lethality, under non-permissive 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 that 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 that result in greatly reduced ability to transport thiamine. 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.

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.

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.

Thiamine pyrophosphate-requiring enzymes are altered during pyrithiamine-induced thiamine deficiency in cultured human lymphoblasts.

The most common of the severe complications of thiamine deficiency are beriberi and Wernicke-Korsakoff syndrome. To help clarify the biochemical basis for these disorders, a cell culture system has been established in which pyrithiamine, a potent thiamine transport inhibitor, was used to mimic different degrees of thiamine deficiency within human lymphoblasts. Activities of both transketolase and alpha-ketoglutarate dehydrogenase (alpha-KGDH) decreased at the same rate and to roughly the same levels in response to thiamine deficiency within a given cell line. However, variation in sensitivity to thiamine deficiency, as judged by the relative percentage of loss of enzymatic activities, was found when different cell lines were compared. When exogenous thiamine pyrophosphate was added to the activity assays, differences between transketolase and alpha-KGDH became readily apparent. Only 25% of the lost transketolase activity was present as apo-enzyme, whereas 70% of the lost alpha-KGDH activity was present in the apo-enzyme form. For transketolase, the non-recoverable activity was due mainly to a decrease in the synthesis rate of the protein during thiamine deficiency, suggesting that thiamine has a direct effect on the expression of the transketolase gene and/or protein.

Molecular genetics of transketolase in the pathogenesis of the Wernicke-Korsakoff syndrome.

Thiamine deficiency, a frequent complication of alcoholism, plays an important role in the pathogenesis of the Wernicke-Korsakoff syndrome [WKS]. Previous work by a number of investigators has implicated the thiamine-utilizing enzyme transketolase [Tk] as being involved mechanistically in the genetic predisposition to WKS. In particular, Tk derived from fibroblasts has been found to have an increased Km app for its cofactor thiamine pyrophosphate [TPP] and/or exist in different isoelectric forms in alcoholic patients with WKS as compared with unaffected individuals. We have demonstrated that these differences are not due to different Tk alleles, tissue-specific Tk isozymes, or differential mRNA splicing. These findings point to other mechanisms to explain the biochemical Tk variants, such as differences in assembly of the functional holoenzyme or differences in modification of the primary translation product. Tk assembly or modification, once biochemically characterized, may be found to be subject to genetic variation.

The thiamine-dependent hysteretic behavior of human transketolase: implications for thiamine deficiency.

We have investigated the hysteretic properties of human transketolase with emphasis on its dependency on thiamine pyrophosphate concentration. As demonstrated previously, the reaction progress curves revealed a slow transition from an initial low velocity to a faster final steady-state velocity, characterized by the rate constant tau-1. The rate of the transition was dependent on the concentration of the thiamine pyrophosphate cofactor, with progressively longer transition times found as the concentration of thiamine pyrophosphate was decreased. At physiological thiamine pyrophosphate concentrations, the inverse rate constant was in the range of 10 to 20 min for fibroblast-derived transketolase and increased dramatically with only small decreases from these levels of thiamine pyrophosphate. Variation in the lag was found when transketolase from different individuals was examined. Moreover, at low levels of thiamine, the rate of the transition was different between fibroblast- and lymphoblast-derived transketolase. The substantial lag in formation of active holoenzyme and the findings of interindividual variation and cell type variation in the lag period suggest mechanisms for the loss of transketolase activity during thiamine deficiency and may explain, at least in part, the differential sensitivity to deficiency demonstrated by tissues and individuals.

Thiamine utilization in the pathogenesis of alcohol-induced brain damage.

There is increasing evidence for the role of thiamine deficiency in ethanol neurotoxicity and in development of alcoholic organic brain disorders other than Wernicke-Korsakoff syndrome [WKS] and cerebellar degeneration. Investigations in humans and in animal models have implicated a reduction in the activities of thiamine-utilizing enzymes as the metabolic basis of tissue injury due to thiamine deficiency. We have investigated the interactions of the thiamine-utilizing enzyme transketolase [Tk], derived from human fibroblasts, lymphoblasts, and various brain regions, with its cofactor, thiamine pyrophosphate [TPP], in an attempt to elucidate the molecular basis of selective brain damage in alcoholism-associated thiamine deficiency. There were no significant differences in the isoelectric pattern of Tk among the nine brain regions (white matter and grey matter) examined. However, Tk activity/mg protein, increase in Tk activity with addition of excess TPP (TPP effect), and TPP-dependent rate of formation of active Tk holoenzyme (tau) varied 2.5-, 6-, and 4-fold, respectively, among these brain regions. These differences in tissue requirements for TPP may contribute to the selective vulnerability of certain brain regions to alcoholism-associated thiamine deficiency, and may influence the pattern of clinical impairment in the individual patient.

Redundant regulatory elements account for the developmental control of a ribosomal protein gene of Dictyostelium discoideum.

In Dictyostelium discoideum, ribosomal protein genes along with other growth specific genes appear to be coordinately regulated, primarily in response to differences in the translational capacity of developing versus growing cells. In particular, expression of the members of this large class of genes is rapidly and dramatically deactivated when the developmental program is initiated and growth and division cease. In order to understand the mechanisms behind the deactivation event and how it is coupled to the transition from growth to development, we have analyzed the promoter of the V18 gene, a ribosomal protein gene characteristic of this class of growth specific genes. We have delineated three discrete regions involved in the transcription and regulation of the V18 gene. A initiator region which appears to function in a TATA-independent manner was required for transcription and for establishing start site utilization. Two regions upstream of this were defined, both of which were found to independently confer proper developmental regulation.

Nutrient-responsive promoter elements of the V4 gene of Dictyostelium discoideum.

Expression of the V4 gene of Dictyostelium discoideum is required for the transition from growth to development in response to an altered nutrient environment. In addition, the expression itself is sensitive to the types and amounts of nutrients supporting growth. We describe the structure of the two copies of the V4 gene and the relationship between these genes and the two V4 mRNA species produced during growth. In addition, three regions were identified within the upstream sequences of the V4b gene that are important for proper transcription. At least two of the regions can, independently of the others, confer deactivation of transcription upon initiation of development and thus serve as redundant regulatory sequences. However, the regions are differentially responsive to the types and amounts of nutrients present in the cell's environment and thus are distinct from one another.

Cloning of human transketolase cDNAs and comparison of the nucleotide sequence of the coding region in Wernicke-Korsakoff and non-Wernicke-Korsakoff individuals.

Variants of the enzyme transketolase which possess reduced affinity for its cofactor thiamine pyrophosphate (high apparent Km) have been described in chronic alcoholic patients with Wernicke-Korsakoff syndrome. Since the syndrome has been shown to be directly related to thiamine deficiency, it has been hypothesized that such transketolase variants may represent a genetic predisposition to the development of this syndrome. To test this hypothesis, human transketolase cDNA clones were isolated, and their nucleotide and predicted amino acid sequence were determined. Transketolase was found to be a single copy gene which produces a single mRNA of approximately 2100 nucleotides. Additionally, the nucleotide sequence of the transketolase coding region in fibroblasts derived from two Wernicke-Korsakoff (WK) patients was compared to that of two nonalcoholic controls. Although nucleotide and predicted amino acid differences were detected between fibroblast cultures and the original cDNAs and among the cultures themselves, no specific nucleotide variations, which would encode a variant amino acid sequence, were associated exclusively with the coding region from WK patients. Thus, allelic variants of the transketolase gene cannot account for the biochemically distinct forms of the enzyme found in these patients nor be considered as a mechanism for genetic predisposition to the development of Wernicke-Korsakoff syndrome. Instead, the underlying mechanism must be extragenic and may be a result of differences in post-translational processing/modification of the transketolase polypeptide.

V4, a gene required for the transition from growth to development in Dictyostelium discoideum.

The V4 gene of Dictyostelium discoideum is regulated in a nutrient-dependent manner and is deactivated immediately upon the onset of development. V4 is expressed only during growth, but its expression is not required for growth. We propose that the V4 gene product plays a role in the transition from growth to development. We have tested this hypothesis by antisense mutagenesis. Cells transformed with a V4 antisense construct contained no detectable endogenous V4 mRNA. These cells grew normally, but they failed to aggregate. Under conditions which normally promote development, V4 antisense transformants failed to deactivate vegetative-specific genes. These cells also were unable to induce the expression of the cAMP cell surface receptor, the cyclic nucleic phosphodiesterase, and contact sites A, all of which are normally induced under such conditions. Surprisingly, cells transformed with a V4 sense construct displayed a similar morphological and biochemical phenotype as the antisense cells, whereas cells transformed with the parental vector exhibited a normal biochemical and morphological phenotype. These results demonstrate that expression of the V4 gene during growth is required for the proper initiation of development.

Structure, expression, and regulation of members of the developmentally controlled V and H gene classes from Dictyostelium.

We have examined the expression and structure of vegetative specific genes belonging to the V and H gene classes. Both classes of genes are deactivated at the onset of development by a reduction in the rate of transcription. Thus, the genes must be reactivated when the terminally differentiated spores germinate and the resulting amebae return to the vegetative state. During germination, activation of expression of most members of the V gene class was found to parallel the emergence of amoebae from the spore coats. The activation of the V genes did not occur when protein synthesis was inhibited. The timing of activation of the H genes was more heterogeneous and did not parallel emergence. H gene activation occurred even when protein synthesis was inhibited. V4 was found to be the only vegetative specific gene that was responsive to the presence of bacteria. V4 expression was induced by 25-100 fold via transcriptional activation when bacteria were added to amebae growing axenically. Isolation and sequence analysis of the corresponding genomic clones revealed that two V genes, V18 and V1, encode ribosomal proteins. Promoter analysis has delineated the sequences necessary for expression and regulation for several of the V and H genes. In all cases, expression was determined by sequences within the first several hundred base pairs of the transcription start site. For V18 and V14, a positive constitutive element was identified in addition to the sequences involved in regulation. Finally, all of the characterizations and findings are discussed in terms of postulated models for V and H gene expression and regulation.

Primary structure and regulation of vegetative specific genes of Dictyostelium discoideum.

We have examined the expression and structure of several genes belonging to two classes of vegetative specific genes of the simple eukaryote, Dictyostelium discoideum. In amebae grown on bacteria, deactivation of all vegetative specific genes occurred at the onset of development and very little mRNA exists by 8 to 10 hours. In contrast, when cells were grown in axenic broth, the mRNA levels remained constant until a dramatic drop occurred around 10 to 12 hours. Thus, regulation of both classes of genes during the first several hours of development is dependent upon the prior growth conditions. Analysis of genomic clones has resulted in the identification of two V genes, V1 and V18, as ribosomal protein genes. Several other V genes were not found to be ribosomal protein genes, suggesting that in Dictyostelium non-ribosomal protein genes may be coordinately regulated with the ribosomal protein genes. Finally, using deletion analysis we show that the promoters of two of the V genes are composed of a constitutive positive element(s) located upstream of sequences involved in the regulated expression of these genes and within the first 545 upstream bp for V18 and 850 bp for V14. The regions involved in regulated expression were localized between -7 and -222 for V18 and -70 and -368 for V14. The sequences conferring protein synthesis sensitivity were shown to reside between -502 and -61 of the H4 promoter.