Propuesta de solución al problema Multimedia soporte digital

Since the discovery of glyoxalase activity a century ago it has been known that the glyoxalase system catalyses the conversion of methylglyoxal to lactate (Dakin and Dudley, 1913). However, this was originally thought to be L-lactate and act as a major metabolic process converting glucose to L-lactate, as discussed by Thornalley (1990). The widespread distribution of glyoxalase in all studied organisms indicated that this enzymatic system performs an important cellular function (Hopkins and Morgan, 1945). Racker identified that metabolism of methylglyoxal to lactate by the glyoxalase system occurred in a two-step process via

60 the intermediate S-D-lactoylglutathione (Racker, 1951). In 1977 it was discovered that methylglyoxal and related aldehydes react with arginine residues, although the physiological significance of this was not understood (Takahashi, 1977). A number of studies in the 1980s and 1990s began to implicate the glyoxalase system and dicarbonyl metabolism in diabetes. An increase in dicarbonyl formation was shown in both hyperglycaemia and in patients with diabetes as well as overexpression of glyoxalase 1 (Glo1) preventing the increase in AGE formation in hyperglycaemia (Thornalley, 1988; Thornalley, et al., 1989). Methylglyoxal was shown to be a protein glycating agent and MG-H1 a significant type of proteome damage and a significant AGE occurring in diabetes (Ahmed, et al., 2005a; Lo, et al., 1994; Thornalley, et al., 2003). Overexpression of Glo1 in endothelial cells has been shown to prevent the increases in MG and associated AGEs observed in hyperglycaemia and to correct the cell dysfunction observed in diabetes (Shinohara, et al., 1998). Recent studies have continued to show the impact of metabolism by the glyoxalase system and alterations therein in diabetes – for example methylglyoxal has been shown to be decreased by the glucose lowering drug metformin, and MG-H1 modification of both collagen IV and LDL has been shown to have functional impairments significant in states of diabetes (Beisswenger, et al., 1999; Dobler, et al., 2006; Rabbani, et al., 2011). Knockdown of Glo1 in mice, leading to a 45 - 65% decrease in the tissue Glo1 activity, resulted in an increase in proteasomal modification and impaired proteasomal activity (Queisser, et al., 2010). In addition, treatment of rodents with a cell permeable fusion protein construct of Glo1 has been shown to prevent the beta cell ablation induced by Streptozotocin treatment, indicative of its protective capacity (Kim, et al., 2013).

2.4.1.2. Glyoxalase pathway

The glyoxalase system consists of two cytosolic enzymes – Glo1 and glyoxalase 2 (Glo2) - which catalyse the conversion of methylglyoxal to D-lactate via the intermediate S-D-lactoylglutathione. It thereby achieves metabolism of methylglyoxal, a detoxification function. GSH binds non-enzymatically to methylglyoxal forming the methylglyoxal-glutathione hemithioacetal, and Glo1 then subsequently catalyses the isomerisation of the hemithioacetal to S-D- lactoylglutathione (Thornalley, 2003a). Glo2 is a thiolesterase which hydrolyses S-

61 D-lactoylglutathione to D-lactate, reforming GSH (Rabbani and Thornalley, 2012a; Thornalley, 1994). These sequential enzymatic reactions are illustrated in Figure 7.

Figure 7: The glyoxalase system. Adapted from Xue, et al. (2011).

Glyoxalase 1

Human glyoxalase 1 is a dimeric Zn2+-metalloenzyme, with one zinc ion per subunit, of molecular mass 42 kDa (Thornalley, 2003a). The translation product of the gene consists of 184 amino acids, of which the amino acid residues tryptophan, lysine, tyrosine, histidine and glutamic acid lie within the active site situated at the dimer interface (Thornalley, 1993; Thornalley, 2003a). Expression of Glo1 as a dimer has been reported in humans, other mammalian species, bacteria and plants (Xue, et al., 2011). In yeast, however, Glo1 is a monomer, of size 32 and 37 kDa in Saccharomyces cerevisae and Schizosaccharomyces pombe, respectively (Xue, et al., 2011).

The human gene, GLO1, has three phenotypes – GLO 1-1, GLO 1-2 and GLO 2-2, representing the homozygous and heterozygous expression of the diallelic gene (Xue, et al., 2011). GLO2 is the ancestral gene and GLO1 is thought to have arisen through mutation (Xue, et al., 2011). The two alleles differ only in the amino acid at position 111 – an alanine in one allele and glutamic acid in the alternative allele (Kim, et al., 1995). The diallelic gene is located on chromosome 6 in humans

62 and on chromosome 17 in mice, the locus of both are closely linked to the major histocompatability complex indicating conservation of linkage groups throughout evolution (Bender and Grzeschik, 1976; Minna, et al., 1978).

Glyoxalase 2

Glyoxalase 2 is a monomeric thiolesterase found in the cytosol of all eukaryotic and prokaryotic organisms (Thornalley, 1993). It molecular mass is ca. 29.5 and 29.0 kDa in mouse and human, respectively, estimated by SDS polyacrylamide gel electrophoresis (Oray and Norton, 1980; Thornalley, 1993). Glo2 contains a Fe(II)Zn(II) centre, with substrate hydrolysis linked to the Zn(II) site (Limphong, et al., 2009). It is a basic protein which is competitively inhibited by the hemithioacetal substrate of Glo1 (Oray and Norton, 1980). Arginine, histidine and lysine residues are present in the Glo2 active site, with histidine important to the enzyme catalysed mechanism (Thornalley, 1990).

The gene for human glyoxalase 2 is hydroxyacylglutathione hydrolase, HAGH, or GLO2. It is located on human chromosome 16 and on chromosome 17 in mice (Tan and Whitney, 1993). There are no common polymorphisms for GLO2 (Thornalley, 1993). A rare polymorphism for GLO2 has been found in Micronesian and Japanese populations (Sugita and Takahama, 1983; Tan and Whitney, 1993; Thornalley, 1993).

S-D-Lactoylglutathione

S-D-Lactoylglutathione is formed from the catalytic action of Glo1 and is hydrolysed by Glo2 to form D-lactate. Addition of S-D-lactoylglutathione to cultures of HL60 cells caused growth arrest and toxicity (Thornalley and Tisdale, 1988). Its formation has been shown to be elevated in hyperglycaemia and clinical complications of diabetes correlated with maintenance of higher cellular levels of S- D-lactoylglutathione (Thornalley, et al., 1989). The concentration of S-D- lactoylglutathione in human blood is ca. 41 nmol per mL red blood cells, increasing to ca. 54 nmol per mL red blood cells in diabetic patients (Thornalley, 1993). It has poor membrane permeability and therefore it is expected that it is produced and hydrolysed within the cytosol (Rae, et al., 1991).

63

D-Lactate

D-Lactate is produced by the thiolesterase Glo2. In physiological systems it is metabolised to pyruvate by the enzyme 2-hydroxyacid dehydrogenase (Thornalley, 1993). However, it is not metabolised by red blood cells (Thornalley, 1988). Activity of this enzyme is lacking in some immortalised cell lines, and therefore D- lactate accumulates. The concentration of D-lactate is ca. 10 and 20 nmol per g blood in healthy controls and diabetic patients, respectively (Thornalley, 1993). Increases in plasma D-lactate concentration are also observed after a meal and after exercise (Kondoh, et al., 1992; Ohmori and Iwamoto, 1988). D-Lactate is membrane permeable, crossing membranes via a specific lactate transporter, the inorganic anion exchanger and by diffusion. Approximately half the D-lactate membrane transport in human red blood cells is via the lactate transporter (Thornalley, 1993).