Taspase represents a highly unique enzyme
Taspase1 represents a highly unique enzyme that has co-evolved in vertebrates and invertebrates together with proteins of the Trithorax/MLL family. The crystal structure of Taspase1 has already been revealed (PDB-ID: 2A8I, 2A8J, 2A8J) (Khan et al., 2005). According to these structures, two monomeric Taspase1 proenzymes (p50) form a homodimer (p50/p50) that are subsequently processed into two α- (amino acids 1–233; p28) and two β-subunits (234–420; p22), both of which remain stably associated (αββα). Importantly, the β-subunit carries the N-terminal threonine-234 residue (T234), which represents the catalytic center (Khan et al., 2005). Thus, a Taspase1 homodimer exhibits two catalytic centers that are oriented in an angle of about 108° at opposing sides of the dimer. Both catalytic centers contain a single chloride ion that is part of an intrinsic regulatory mechanism and inhibits Taspase1 at physiological sodium chloride concentrations (IC50~25mM NaCl) (Khan et al., 2005; Michalska et al., 2006). The target consensus sequence hydrolyzed by Taspase1 is Q3[F,I,L,V]2D1 G1\'X2\'X3\'D4\', but only MLL, MLL4 and TFIIA as well as the oncogenic AF4–MLL fusion protein are confirmed substrates (Bursen et al., 2004; Hsieh et al., 2003b; Zhou et al., 2006).
For the AF4–MLL fusion protein the Taspase1-induced protein fragments (p178N and p180C) heterodimerize via specific domains localized in both protein fragments (FYRN and FYRC) (Yokoyama et al., 2002; Hsieh et al., 2003a). After dimerization, a multi-protein complex is formed that exerts strong oncogenic activities: a) ectopic activation of P-TEFb kinase results in a hyperactivation of transcriptional dna-pkcs processes, and b) co-bound histone methyltransferases cause ectopic H3K4/H3K79 patterns which subsequently effectuate epigenetic deregulation in a genome-wide fashion (Benedikt et al., 2011). Without the fragment dimerization both AF4–MLL fragments are subject to proteasomal destruction (Pless et al., 2011).
Discussion In the context of t(4;11) leukemia, Taspase1 is a conditional oncoprotein because it mediates the proteolysis of the AF4–MLL fusion protein (p328). The resulting protein fragments (p178N and p180C) form a heterodimer which resembles a molecular platform for the assembly of the highly stable AF4–MLL multiprotein complex. This high molecular weight complex exerts an enhanced P-TEFb activity and causes ectopic histone signatures (Benedikt et al., 2011). Retroviral expression of the AF4–MLL fusion protein in murine hematopoietic stem/precursor cells caused the development of proB ALL in mice (Bursen et al., 2010). Consequently, we decided to investigate Taspase1 at the molecular level, aiming to understand the functional relationship between Taspase1 and AF4–MLL, and to translate this knowledge into novel strategies aiming to block the oncogenic functions deriving from AF4–MLL. First, we refined the available crystal structure of human Taspase1 by in silico modeling and used this structural model of Taspase1 to identify critical amino acid residues. Based on our data, dimerization of two Taspase1 monomers seems to be a key step for activating the intrinsic autoproteolytic function (Fig. 6). There are several important amino acids, but S291 and D233 are critical for the hydrolysis of the peptide bond between residues D233 and T234. This is in line with recent findings obtained for several proteases, demonstrating that a single serine residue is sufficient to cause autoproteolytic activity (Polgár, 2005). The T234 residue apparently remains in an inactive conformation due to the presence of a chloride ion, which is coordinated by the peptide backbone of G49, N100 and T234. This chloride anion functions as a reversible competitive inhibitor at physiological chloride concentrations (IC50~25mM NaCl) (Khan et al., 2005). Binding of a cognate substrate leads to the displacement of the inhibitory chloride anion, possibly by using the carboxyl moiety of D4\' deriving from the consensus cleavage site (Q3[F,I,L,V]2D1 G1\'X2\'X3\'D4\'). As already described by Khan et al. (2005) the displacement of the chloride ion allows T234 to rotate by approximately 180° into the appropriate position to perform the nucleophilic attack at the peptide bond between D1 and G1\' of bound substrate protein. This explains, why many efforts aiming to identify a potent lead which targets the enzymatic center of Taspase1 remained so far without success.