Chapter 1 Introduction and main aims CONTENTS. 1.1 – Branched chain aminotransferase.................................................................... 2 1.2 – hBCAT enzyme kinetics. ................................................................................... 4 1.3 – The structure of the hBCAT proteins. .............................................................. 6 1.4 – Human branched-chain amino acid metabolism. .......................................... 10 1.5 – Redox regulation of hBCAT and relation to other redox proteins. ............... 15 1.6 – hBCAT distribution. ......................................................................................... 19 1.7 – BCAT and the glutamate-glutamine cycle. ..................................................... 21 1.8 – hBCAT and the BCAAs in the brain. ............................................................... 25 1.8.1 – Maple syrup urine disease. .......................................................................... 25 1.8.2 – Excitotoxicity pathway. ................................................................................ 27 1.9 – Alzheimer’s disease. ........................................................................................ 31 1.9.1 – Amyloid pathology. ...................................................................................... 32 1.9.2 – Tau pathology. ............................................................................................. 33 1.9.3 – Protein synthesis and autophagy. ................................................................ 35 1.9.4 – Protein misfolding and redox pathology. ...................................................... 37 1.9.5 – The blood-brain barrier. ............................................................................... 38 1.10 – Aims and objectives. ..................................................................................... 45 1 Chapter 1 – Introduction and main aims. Forshaw, T. E. (2016) 1.1 – Branched chain aminotransferase. Branched chain aminotransferase (BCAT) [E. C. 2.6.1.42] is a highly conserved enzyme essential for mammalian branched chain L-amino acid (BCAA) and branched chain keto acid (BCKA) metabolism (Bledsoe et al., 1997; Ichihara & Koyama, 1966; Schuldiner et al., 1996; Suryawan et al., 1998). The most described function of the BCAT enzymes is to catalyse the reversible transamination of the BCAAs (L-leucine, L-valine and L- isoleucine) and α-ketoglutarate (KG), to generate L-glutamate and the respective BCKAs (α-ketoisocaproate (KIC), α-ketoisovalerate (KIV), and α-keto-β-methylvalerate (KMV) (Scheme 1.1). Rat in vivo studies utilising 15N-labelled L-leucine have determined that the preferred direction of transamination is towards L-glutamate production, although labelled L-isoleucine and L-valine were also detected (Kanamori et al., 1998; Sakai et al., 2004). Specifically, in rat neurons it has been observed that the BCAT enzymes are involved in the de novo synthesis of approximately 30% of brain glutamate (LaNoue et al., 2001). Scheme 1.1 – Transamination catalysed by BCAT. Human BCAT (hBCAT) exists as two isozymes, the cytosolic (hBCATc) and the mitochondrial (hBCATm), which show distinct cellular localisation (Hutson et al., 1988). Human BCATc and hBCATm have a molecular mass of 44.3 kDa and 41.7 kDa, respectively, and their primary amino acid sequence has 395 and 365 amino acids, respectively. These isozymes share 58% amino acid homology, with the greatest homology in the active sites (Davoodi et al., 1998). Both of the full length enzymes have been purified, first from rat tissue (Hall et al., 1993), and later the human protein from cloned genes overexpressed in E. coli (Davoodi et al., 1998). This has allowed for detailed kinetic and redox characterisation, as well as solving of the crystal structures (Conway et al., 2002; Conway et al., 2008; Goto et al., 2005; Yennawar et al., 2001). 2 Chapter 1 – Introduction and main aims. Forshaw, T. E. (2016) As with all aminotransferases, hBCAT uses a pyridoxal phosphate (PLP) co-factor which is bound to an active site L-lysine residue. The PLP-dependent enzyme family is divided into five different ‘fold types’ by the specific orientation of PLP within the active site (Reviewed by Eliot & Kirsch, 2004). While most mammalian aminotransferases belong to fold type I, hBCAT is the only mammalian type IV aminotransferase (Yennawar et al., 2001; Yennawar et al., 2002). The other fold type IV enzymes are bacterial D-amino acid aminotransferases (Martínez del Pozo et al., 1989); differing significantly from the type I and II in that the active site has a mirror image orientation (Yennawar et al., 2001). In fold type IV enzymes, the active site L-lysine residue is bound to PLP on the re face of the chiral centre, exposing the si face to solvent, while the opposite is true for type I aminotransferases (Sugio et al., 1995). This suggests that hBCAT may have a different role in mammals from that of other aminotransferases. The genes for hBCATc and hBCATm are BCAT1 and BCAT2, respectively. Both genes can be transcribed by activating transcription factor 4 (ATF4) and hBCATm alone by C/EBP homologous protein (CHOP), in response to cellular stresses such as starvation, misfolded proteins, and oxidative stress (Harding et al., 2003; Han et al., 2013). Additionally, the BCAT1 gene may be promoted by c-myc, which is involved in cell cycle progression and apoptosis (Ben-Yosef et al., 1998) and the gene has been described as an oncogene (Zhou et al., 2013; Tönjes et al., 2013). There are five variants of BCAT1 mRNA, however, four of these have only been observed in cDNA arrays and only the longest variant is observed as hBCATc protein (Ota et al., 2004; Bechtel et al., 2007). Likewise, the longest transcript of the BCAT2 gene translates to the most widely observed hBCATm protein (Than et al., 2001). Unlike hBCATc, the BCAT2 gene has been obsevered to be transcribed by Kruppel-Like factor 15 (Klf15), a glucocorticoid receptor target (Kuo et al., 2013; Shimizu et al., 2011). There are two additional splice variants of BCAT2 (Than et al., 2001; Lin et al., 2001). The first, placental protein 18b (PP18b), is weakly expressed in many tissues, although its function is not clearly understood (Than et al., 2001). The variant protein has a molecular weight 3 Chapter 1 – Introduction and main aims. Forshaw, T. E. (2016) approximately 8 kDa lighter than hBCATm and lacks the N-terminal mitochondrial targeting sequence, resulting in cytoplasmic localisation (Than et al., 2001). The second transcript, termed P3, was identified as an inhibitor of thyroid hormone receptor (TR) transcriptional activity (Lin et al., 2001). This varient localises to both the mitochondria and nucleus of cells, acting both as a TR antagonist and as an inhibitor of TR binding to DNA. This suggests that hBCATm may control expression of other proteins, although P3 is currently the only isoform published as demonstrating this activity. 1.2 – hBCAT enzyme kinetics. Characterisation of hBCAT has demonstrated that the enzymes both follow the common aminotransferase ‘ping pong bi bi’ mechanism for multi-substrate enzymes, as described by Cleland (1963). For this it is a requirement that PLP is bound as a Schiff base to the Ɛ-amine of active site lysine (Lys202 for hBCATm and Lys222 for hBCATc) to form an enzyme-cofactor complex (E-PLP). The first step in catalysis is the modification of the complex by transfer of an amine group from a substrate L-amino acid to PLP, forming pyridoxamine phosphate (PMP) and releasing an α-keto acid as a first product (Figure 1.1). In the next step, a substrate α-keto acid abstracts the amino group from PMP in a reaction catalysed by the protein, restoring the molecule to the native PLP form and releasing an amino acid product (Figure 1.1). Through both steps the cofactor will disassociate and form external aldimine and ketimine intermediates with the substrate, before reassociation as an E-PLP or enzyme-PMP (E-PMP) complex (Yennawar et al., 2002). In addition to the earlier described substrates, the straight chain analogues of L- valine (norvaline) and L-leucine (norleucine) and the α-keto acid conjugate of methionine (α-keto-γ-methiobutyrate) are substrates for hBCAT. However, neither of D-isoleucine, the aromatic essential L-amino acids L-alanine or L-aspartate were active substrates (Davoodi et al., 1998). Kinetic studies of the hBCAT enzymes have determined that the first step of transamination is the slower rate determining step, while the second step has 4 Chapter 1 – Introduction and main a ims . Forshaw, T. (2016) E. Figure 1.1 – Ping-pong kinetics of the BCAT enzymes. The timeline illustrates the course of transamination from amino acid to α-keto acid. Starting from the left side, the amine group from an amino acid is transferred to enzyme-PLP complex to form PMP and an α-keto acid. The α- keto acid is then released and an enzyme-PMP complex is formed. An α-keto acid may then react with the complex and abstract the amino group from PMP, resulting in release of amino acid and restoration of the enzyme-PLP complex. Abbreviations: PLP – co-factor. PMP – amine modified co-factor. E-PLP/PMP – complex of enzyme and co-factor. BCAA – branched-chain amino acid. Glu – L-glutamate. 5 Chapter 1 – Introduction and main aims. Forshaw, T. E. (2016) a turnover rate approximately double that of the first (Table 1.1). Further characterisation by Yennawar et al. (2006) determined that the equilibrium constant (Keq) for each of the