Transaldolase (TAL) is an enzyme of the pentose phosphate pathway (PPP) which generates two essential metabolites: ribose 5-phosphate for nucleotide synthesis and NADPH for biosynthetic reactions and maintenance of glutathione (GSH) at a reduced state to protect sulfhydryl groups and cellular integrity from oxygen radicals. We have determined that changes in TAL expression and enzymatic activity significantly impact the overall function of the PPP and susceptibility to apoptosis signals. Overexpression of TAL in Jurkat and H9 human T cells was accompanied by a decrease in glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities, a concomitant depletion in NADPH and GSH levels, and increased sensitivity to apoptosis provoked by serum deprivation, H202, NO, TNFca Fas antibody, or infection by HTV-1. In contrast, suppression of TAL activity increased G6PD and 6PGD activities, augmented GSH levels, and inhibited these apoptosis pathways. TAL regulates key checkpoints of apoptosis: formation of reactive oxygen intermediates (ROI), mitochondrial permeability transition, activation of caspases, phosphatidylserine externalization, and ultimately, cell death. Thus, TAL serves as a critical determinant of tissue and cell type-specific functioning of the PPP and sensitivity to apoptotic signals. This is consistent with the importance of TAL in determining systemic behavior and propagation of biochemical signals within a metabolic network. The long-term goal of this proposal is to delineate transcriptional and postranslational determinants that regulate tissue- and cell-type specific activity of TAL and its effect on the PPP and sensitivity to apoptosis signals. We have identified two distinct transcriptional regulatory elements, an upstream promoter and an enhancer, the latter contributed by a TAL-associated repetitive element (TARE). Boundaries of promoter and enhancer elements will be further defined by transfection, gel retardation, and footprinting studies. TAL-H is stoichiometrically phosphorylated and its enzymatic activity is stimulated by PKC. Phosphorylation sites will be delineated by phosphopeptide mapping and site-directed mutagenesis. Their impact on TAL activity will be determined using in vitro and in vivo kinase and transfection assays. To better define the mechanisms of TAL-induced changes on functioning of the PPP, levels of PPP metabolites and enzyme activities will be assessed in cells with altered TAL activities. The proposed studies with the availability of unique reagents and existing model systems will further our knowledge on the role of TAL in the PPP, apoptosis signaling, and related disease processes.
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