First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target.

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.

Key glycolytic branch influences mesocarp oil content in oil palm.

The fructose-1,6-bisphosphate aldolase catalyzed glycolysis branch that forms dihydroxyacetone phosphate and glyceraldehyde-3-phosphate was identified as a key driver of increased oil synthesis in oil palm and was validated in Saccharomyces cerevisiae. Reduction in triose phosphate isomerase (TPI) activity in a yeast knockdown mutant resulted in 19% increase in lipid content, while yeast strains overexpressing oil palm fructose-1,6-bisphosphate aldolase (EgFBA) and glycerol-3-phosphate dehydrogenase (EgG3PDH) showed increased lipid content by 16% and 21%, respectively. Genetic association analysis on oil palm SNPs of EgTPI SD_SNP_000035801 and EgGAPDH SD_SNP_000041011 showed that palms harboring homozygous GG in EgTPI and heterozygous AG in EgGAPDH exhibited higher mesocarp oil content based on dry weight. In addition, AG genotype of the SNP of EgG3PDH SD_SNP_000008411 was associated with higher mean mesocarp oil content, whereas GG genotype of the EgFBA SNP SD_SNP_000007765 was favourable. Additive effects were observed with a combination of favourable alleles in TPI and FBA in Nigerian x AVROS population (family F7) with highest allele frequency GG.GG being associated with a mean increase of 3.77% (p value = 2.3E-16) oil content over the Family 1. An analogous effect was observed in yeast, where overexpressed EgFBA in TPI - resulted in a 30% oil increment. These results provide insights into flux balances in glycolysis leading to higher yield in mesocarp oil-producing fruit.

Aging-related changes of triose phosphate isomerase in hippocampus of senescence accelerated mouse and the intervention of acupuncture.

Glycometabolism disorder induced by triose phosphate isomerase (TPI) is closely related to Alzheimer's disease (AD). Senescence accelerated mouse (SAM) is often employed as an AD model characteristic of early cognitive impairment. In order to investigate the variation of TPI with aging, SAM prone 8 (SAMP8) and SAM resistant 1 (SAMR1) were divided into 2-month, 6-month, 8-month and 12-month group. For the analysis of acupuncture intervention, SAMP8 were divided into SAMP8 control group (Pc), SAMP8 acupoint group (Pa), SAMP8 non-acupoint group (Pn) and SAMR1 control group (Rc). Grading score of senescence and Morris water maze results showed that SAMP8 presented aging-related deterioration of learning and memory, and that acupuncture could improve the learning and memory ability of SAMP8. TPI activity and expression were detected by colorimetric method and Western blot analysis, respectively. When compared to SAMR1, TPI activity in 6-, 8- and 12-month SAMP8 decreased significantly. However, acupuncture intervention markedly up-regulated TPI activity in hippocampus of Pa. These findings suggested that the learning and memory deterioration of SAMP8 with aging might be associated with the lower TPI activity and that acupuncture could improve the cognitive impairment by increasing TPI activity, thus correcting the abnormal glycolysis metabolism and maintaining the brain homeostasis and internal environment.

Analyzing the effect of decreasing cytosolic triosephosphate isomerase on Solanum tuberosum hairy root cells using a kinetic-metabolic model.

A kinetic-metabolic model of Solanum tuberosum hairy roots is presented in the interest of understanding the effect on the plant cell metabolism of a 90% decrease in cytosolic triosephosphate isomerase (cTPI, EC 5.3.1.1) expression by antisense RNA. The model considers major metabolic pathways including glycolysis, pentose phosphate pathway, and TCA cycle, as well as anabolic reactions leading to lipids, nucleic acids, amino acids, and structural hexoses synthesis. Measurements were taken from shake flask cultures for six extracellular nutrients (sucrose, fructose, glucose, ammonia, nitrate, and inorganic phosphate) and 15 intracellular compounds including sugar phosphates (G6P, F6P, R5P, E4P) and organic acids (PYR, aKG, SUCC, FUM, MAL) and the six nutrients. From model simulations and experimental data it can be noted that plant cell metabolism redistributes metabolic fluxes to compensate for the cTPI decrease, leading to modifications in metabolites levels. Antisense roots showed increased exchanges between the pentose phosphate pathway and the glycolysis, an increased oxygen uptake and growth rate.

A large decrease of cytosolic triosephosphate isomerase in transgenic potato roots affects the distribution of carbon in primary metabolism.

Triosephosphate isomerase (TPI, EC 5.3.1.1) catalyzes the interconversion of dihydroxyacetone-P and glyceraldehyde 3-P in the glycolytic pathway. A constitutively expressed antisense construct for cytosolic TPI was introduced into potato (Solanum tuberosum) using Agrobacterium rhizogenes to examine the metabolic effects of a reduction in cytosolic TPI in roots. We obtained a population of transgenic root clones displaying ~36 to 100 % of the TPI activity found in control clones carrying an empty binary vector. Ion exchange chromatography and immunoblot analysis showed that the antisense strategy significantly decreased the cytosolic TPI isoform, while levels of plastidial TPI activity remained apparently unaffected. Transgenic roots were characterized with respect to the activity of glycolytic enzymes, their metabolite contents and carbon fluxes. Metabolite profiling of sugars, organic acids, amino acids and lipids showed elevated levels of sucrose, glucose, fructose, fumarate, isocitrate, 4-aminobutyrate, alanine, glycine, aromatic amino acids and saturated long chain fatty acids in roots containing the lowest TPI activity. Labelings with (14)C-glucose, (14)C-sucrose and (14)C-acetate indicated that a reduction of cytosolic TPI activity in roots increased carbon metabolism through the pentose phosphate pathway, O(2) uptake and catabolism of sucrose to CO(2), and capacity for lipid synthesis. These results demonstrate that a large reduction of cytosolic TPI alters the distribution of carbon in plant primary metabolism.

Broad distribution of TPI-GAPDH fusion proteins among eukaryotes: evidence for glycolytic reactions in the mitochondrion?

Glycolysis is a central metabolic pathway in eukaryotic and prokaryotic cells. In eukaryotes, the textbook view is that glycolysis occurs in the cytosol. However, fusion proteins comprised of two glycolytic enzymes, triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were found in members of the stramenopiles (diatoms and oomycetes) and shown to possess amino-terminal mitochondrial targeting signals. Here we show that mitochondrial TPI-GAPDH fusion protein genes are widely spread across the known diversity of stramenopiles, including non-photosynthetic species (Bicosoeca sp. and Blastocystis hominis). We also show that TPI-GAPDH fusion genes exist in three cercozoan taxa (Paulinella chromatophora, Thaumatomastix sp. and Mataza hastifera) and an apusozoan protist, Thecamonas trahens. Interestingly, subcellular localization predictions for other glycolytic enzymes in stramenopiles and a cercozoan show that a significant fraction of the glycolytic enzymes in these species have mitochondrial-targeted isoforms. These results suggest that part of the glycolytic pathway occurs inside mitochondria in these organisms, broadening our knowledge of the diversity of mitochondrial metabolism of protists.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation.

Alzheimer's disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Metabolite and enzyme profiles of glycogen metabolism in Methanococcoides methylutens.

When a buffered anaerobic cell suspension of Methanococcoides methylutens was maintained under methanol-limited conditions, intracellular glycogen and hexose phosphates were consumed rapidly and a very small amount of methane formed at 4 h of a starvation period. When methanol was supplemented after a total of 20 h of starvation, a reverse pattern was observed: the glycogen level and the hexose phosphate pool increased, and formation of methane took place after a lag period of 90 min. A considerable amount of methane was formed in 120 min after its detection with a rate of 0.18 micromol mg(-1) protein min(-1). When methane formation decreased after 270 min of incubation and finally came to a halt, probably due to complete assimilation of supplemented methanol, the levels of glycogen and hexose monophosphates decreased once again. However fructose 1,6-diphosphate levels showed a continuous increase even after exhaustion of methane formation. In contrast to the hexose phosphate pool, levels of other metabolites showed a small increase after addition of methanol. The enzyme profile of glycogen metabolism showed relatively high levels of triose phosphate isomerase. Glyceraldehyde 3-phosphate dehydrogenase reacted with NADPH with a three-fold higher activity as compared to that with NADH.

Activating oligomerization as intermediate level of signal transduction: analysis of protein-protein contacts and active sites in several glycolytic enzymes.

A number of enzymes have inactive monomeric and active oligomeric forms. This suggests presence of definite interglobular contact -active site interaction in the enzymes. Although the phenomenon is widely studied in vitro as part of folding process the biological roles of the phenomenon, termed here as "activating oligomerization" are not clearly understood. In this work a procedure for analysis of protein-protein interactions was elaborated. Using spatial structures of several glycolytic enzymes potential role of kinase phosphorylation in regulation of oligomerization of the proteins as well as association of domains in a two-domain protein was assessed. In the enzymes 15-75% of kinase sites (mainly protein kinase C and casein kinase 2 sites) are placed in interglobular contact region(s). Upon being phosphorylated these sites may prevent oligomer formation. In structures of all the enzymes definite evidences of connection between active site and interglobular contact were found. Two structural mechanisms of interglobular contact influence on the active site were proposed. In addition to known mechanism of oligomerization initiated by allosteric metabolites the influence may be also exerted through functional sequence overlap and/or interdomain contact stabilization mechanisms. Implications for regulation of enzyme cellular function(s), signal transduction and metabolic analysis are considered. It is concluded that activating oligomerization may represent an intermediate level of enzyme cellular regulation.