Blockade of enzyme activity inhibits tissue transglutaminase-mediated transamidation of α-synuclein in a cellular model of Parkinson's disease

Transamidation of alpha-synuclein by the Ca2+-dependent enzyme tissue transglutaminase (tTG, EC 2.3.2.13) is implicated in Parkinson's disease (PD). tTG may therefore offer a novel therapeutic target to intervene in PD. Here we first evaluated the potency and efficacy of three recently developed irreversible active-site inhibitors of tTG (B003, Z006 and KCC009) to inhibit tTG activity in vitro and in living cells. In vitro, all compounds were found to be full inhibitors of tTG activity showing a rank order of potency (defined by IC-50 values) of Z006 > B003 > KCC009. Upon Ca2+ ionophore (A23187) induced activation of cellular tTG (measured by incorporation of the tTG-specific amine substrate 5-(biotinamido)pentylamine (BAP) into cellular proteins) in neuroblastoma SH-SY5Y cells, only Z006 (0.3-30 mu M) retained the capacity to completely inhibit tTG activity. Under these conditions B003 (3-300 mu M) only partially blocked tTG activity whereas KCC009 (3-100 mu M) failed to affect tTG activity at any of the concentrations used. Z006 (30 mu M) also blocked the tTG mediated incorporation of BAP into alpha-synuclein monomers and SDS-resistant multimers in vitro and in alpha-synuclein overexpressing SHSY5Y cells exposed to A23187 or the PD mimetic 1-methyl-4-phenylpyridine (MPP+). Moreover, Z006 (30 mu M) substantially reduced formation of SDS-resistant alpha-synuclein multimers in SH-SY5Y cells exposed to A23187 or MPP+. in the absence of BAP. We conclude that alpha-synuclein is a cellular substrate for tTG under conditions mimicking PD and blockade of tTG activity counteracts alpha-synuclein transamidation and aggregation in vitro and in living cells. Moreover, our cell model appears an excellent readout to identify candidate inhibitors of intracellular tTG. (C) 2011 Elsevier Ltd. All rights reserved.