IDH1 (Isocitrate Dehydrogenase 1)
The IDH1 gene on chromosome 2 encodes the cytosolic isoenzyme of isocitrate dehydrogenase, enzyme that catalyzes the oxidative decarboxylation of isocitrate into alpha-ketoglutarate (alpha-KG). Hogeboom and Schneider were researching the intracellular distribution of the enzymes responsible for the reactions of the Krebs tricarboxylic cycle when they first reported this enzyme in cytosol in 1950 (Hogeboom and Schneider 1950). Chen and collaborators found that IDH1 was consistent with a dimeric molecular structure, that the gene locus is probably autosomal, and it is different from the mitochondrial locus of isocitrate dehydrogenase (Chen et al. 1972). The single subunit of the enzyme is 414 amino acids long with an estimated molecular mass of 46.65 kDa and contains a C-terminal tripeptide, alanine-lysine-leucine, which is the type 1 peroxisomal targeting sequence (Nekrutenko et al. 1998). IDH1 is involved in many cellular processes such as maintaining cellular cholesterol and fatty acid homeostasis, participating in the redox balance, and in beta-cells it participates in the control of pancreatic glucose-stimulated insulin secretion (Lee et al. 2002; Ronnebaum et al. 2006; Shechter et al. 2003). Approximately 27% of the total IDH1 is associated with peroxisome that is the only NADPH provider (Geisbrecht and Gould 1999).
A gene expression analysis in 22 glioblastoma multiforme samples presented 12% of mutation on the IDH1 gene and was associated with a prolonged patient survival (Parsons et al. 2008). This mutation was also found in acute myeloid leukemia, chondrosarcoma, intrahepatic cholangiocarcinoma, and angioimmunoblastic T-cell lymphoma patients (Molenaar et al. 2014). Since then, this mutation has been studied as a prognostic marker as well as a therapeutic target.
Several studies have been comparing the activity and function of the cytosolic NADP+-dependent isocitrate dehydrogenase between different kingdoms. The IDH proteins seem to have a high degree of amino acid conservation. There are 157 identical amino acid positions among all eukaryotic IDH proteins, and 152 of these are also identical in the Proteobacteria Sphingomonas yanoikuyae polypeptide. One of these conserved amino acid residues are involved in the binding of the isocitrate-Mg2+ complex (Nekrutenko et al. 1998).
Eukaryotic cells express five isocitrate dehydrogenases that catalyze the oxidative decarboxylation of isocitrate into alpha-HG. There are three enzymes that utilize NAD as cofactor (NAD+-dependent isocitrate dehydrogenase), and their location are all in the mitochondrial matrix (Geisbrecht and Gould 1999). Two enzymes utilize NADP as cofactor (NADP+-dependent isocitrate dehydrogenase), one is located in mitochondria (IDH2) and the other is predominantly cytosolic (IDH1) (Geisbrecht and Gould 1999).
In contrast to eukaryotes, Escherichia coli does not present NAD+-dependent isocitrate dehydrogenase and contains only one type NADP+-dependent isocitrate dehydrogenase (Gálvez and Gadal 1995). E. coli and mammals share about 14% of sequence identity of IDH, and its activity is controlled by the phosphorylation/dephosphorylation at a single serine residue (S113), where the phosphorylation prevents the isocitrate binding (Gálvez and Gadal 1995; Nekrutenko et al. 1998). However, the isocitrate dehydrogenase found in the Proteobacteria S. yanoikuyae shows 62% of identity with the human NADP-dependent cytosolic enzyme (Nekrutenko et al. 1998).
All mammalian cytosolic IDH contain a C-terminal tripeptide, alanine-lysine-leucine, which has been identified as a type I peroxisomal targeting signal (PTS1) (Nekrutenko et al. 1998). PTS1 is a tripeptide sequence, which is typically found at the C-terminus of peroxisomal proteins (Nekrutenko et al. 1998). PTS1 has also been described in yeast and some plants (Nekrutenko et al. 1998; Geisbrecht and Gould 1999).
The phylogenetic analysis of the IDH family suggested that the mitochondrial form is more closely related to the cytosolic form of the enzyme than the cytosolic forms between the kingdoms. Then, these genes arose through separate gene duplication events and are not orthologous in animals, fungi, and plants (Nekrutenko et al. 1998). Saccharomyces cerevisiae, classified as members of the fungus kingdom, have 59% identical to human IDH1 (Geisbrecht and Gould 1999).
IDH1 is an asymmetric homodimer in the cytoplasm. The coding regions encode a single subunit of the enzyme that is 414 amino acids long (Parsons et al. 2008). Each monomer consists of three domains: a large domain, a small domain, and a clasp domain.
According to Xu and collaborators, the large (residues 1 – 103 and 286 – 414) and small (104 – 136 and 186 – 285) domains are joined together by a beta-sheet and there are clefts flanked on each side of the beta-sheet, and the active site is localized in the deep cleft and is formed by the large and small domains of one subunit and the small domain of the adjacent subunit (Xu et al. 2004). The active cleft consists of a NADP-binding site and the isocitrate-metal ion-binding site (Parsons et al. 2008). The Arg132 residue forms hydrophilic interactions with the alpha-carboxylate of isocitrate (Parsons et al. 2008). The clasp (residues 137 – 185) domain holds the two subunits together (Xu et al. 2004).
In its structure, the IDH1 features the peroxisomal targeting PST-1 sequence (Ala-Lys-Leu-COOH), a subcellular peroxisomes localization (Shechter et al. 2003).
IDH1 reaction converts isocitrate to alpha-HG and reduces NADP+ to NADPH, and in hypoxic contexts it can make the reverse reaction (Molenaar et al. 2014). IDH1 is the major cytosolic NADPH producer, 13- and 24-fold higher than that of glucose-6-phosphate dehydrogenase and malic enzyme, respectively (Koh et al. 2004). One possible explanation for this is that IDH1 has no negative feedback by NADPH such as the mentioned enzymes above.
NADPH is an essential cofactor for the regeneration of glutathione (GSH), which is an important nonenzymatic antioxidant. GSH is the reduced form, and this thiol group is able to donate a reducing equivalent to other molecules, such as reactive oxygen species to neutralize them, and it becomes the oxidized form glutathione disulfide (GSSG) (Lee et al. 2002). After oxidized to GSSG, GSH levels are restored by NADPH-dependent enzyme activity glutathione reductase (GR), which reduces GSSH to GSH (Jo et al. 2002). Furthermore, NADPH is required for the formation of active catalase tetramers, activity of cytochrome p450, and thioredoxins system (Molenaar et al. 2014). These are all involved with the cellular antioxidant balance. NADPH provided by IDH1 plays a key role in cellular protection against ultraviolet radiation-induced oxidative damage as well (Jo et al. 2002).
Despite its role in protecting against oxidative damage, IDH1 is susceptible to oxidative inactivation by nitric oxide, peroxynitrite, reactive oxygen species (ROS), and lipid peroxidation products (Batinic-Haberle and Benov 2008). Then, enzymes that maintain the cell redox balance are important in protecting IDH1 against inactivation, such as superoxide dismutase (SOD) (Batinic-Haberle and Benov 2008).
NADPH is required for the fat and cholesterol biosynthesis such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA reductase, and 2,4-dienoyl-CoA reductase enzymes. It has been shown that IDH1 activity is positively correlated with the degree of these biosynthesis pathways in mice and human (Koh et al. 2004). Furthermore, the transcription factors SREBP1 e SREBP2 that upregulate the synthesis of several enzymes involved in cholesterol and fatty acid synthesis can activate the IDH1 promoter genes (Shechter et al. 2003). In the peroxisome, IDH1 is the only NADPH supplier. In this organelle, NADPH and alpha-HG is required for the phytanoyl-CoA hydroxylase reaction which is necessary for alpha-oxidation of branched chain fatty acids (Shechter et al. 2003).
Genetics characterization in several cancer types has been carried out looking for new therapeutic targets. In this way, IDH1 mutation was discovered in the glioblastoma multiforme, and now we know it can be present in acute myeloid leukemia, chondrosarcoma, intrahepatic cholangiocarcinoma, and angioimmunoblastic T-cell lymphoma (Molenaar et al. 2014). This mutation is heterozygous and occurs at a single residue of IDH1 in the active site, and it is more frequent at arginine 132 (R132) where R132 is replaced by histidine (R132H), cysteine (R132C), serine (R132S), glycine (R132G), leucine (R132L), or glutamine (R132Q) (Molenaar et al. 2014). The exchange of an amino acid with strong positively charged to lower electrical amino acids results in reorganization of the enzyme active site and appears to favor the active closed state of the enzyme (Molenaar et al. 2014). Consequently, IDH1 mutation leads to a loss of normal enzyme function and confer a neoenzymatic gain of function for NADPH-dependent reduction of alpha-KG to d-2-hydroxyglutarate (2HG) (Dang et al. 2009).
This neoenzymatic function causes several changes in IDH1-mutated cells. The redox balance is affected by 2HG-induced oxidative stress and by decreasing NADPH which is a cofactor required in GSH reaction, a nonenzymatic antioxidant (Reitman and Yan 2010). Moreover, 2HG leads epigenetic dysregulation through competitive inhibitor of multiple alpha-KG-dependent dioxygenases such as TET2, EGLN, and Jumonji-C domain-containing histone demethylases and causing DNA hypermethylation and altered HIF intracellular concentrations, for example (Chan et al. 2015; Molenaar et al. 2014). In acute myeloid leukemia, IDH1 mutation directly inhibits cytochrome c oxidase through 2HG accumulation (Chan et al. 2015). All these alterations can range depending on the mutation in IDH1 since each mutant has a percentage of 2HG production (Molenaar et al. 2014). The most frequent mutation in glioma and acute myeloid leukemia (AML) is the R132H, and this is the weak 2HG producer (Reitman and Yan 2010). R132H is associated with a relatively prolonged patient survival for glioblastoma but not for AML (Molenaar et al. 2014). In this way, just the kind of mutation is not sufficient to define the action of this mutation in cancer. It has been hypothesized that the intracellular 2HG concentration, which gives the largest growth advantage, varies depending on the tumor’s cell type of origin.
In conclusion, several reports provide evidence that IDH1 plays an important role in cell metabolism. This cytosolic NADP+-dependent isocitrate dehydrogenase is the major cytosolic NADPH producer, which is a cofactor in several biological reactions, and is required for regeneration of glutathione, fat, and cholesterol biosynthesis. This IDH isoform also plays a critical role by supplying the cofactor and alpha-KG for the pyruvate cycle between mitochondria and cytosol. In some types of cancer, it was demonstrated that heterozygous residue mutation of IDH1 in the active site leads to a loss of normal enzyme function and confers a neoenzymatic gain of function for NADPH-dependent reduction of alpha-KG to HG. The new function changes cell metabolism, and this is reflected in better or worse patient prognosis depending on the specific mutation in IDH1 and the tumor’s cell type of origin. Further experimental studies are required to improve the understanding of IDH1 mutation in cancer cells, and how tumor types affect the action of this mutation.
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