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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Blockade of enzyme activity inhibits tissue transglutaminase-mediated transamidation of a-synuclein in a cellular model of Parkinson's disease
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Blockade of enzyme activity inhibits tissue transglutaminase-mediated transamidation of a-synuclein in a cellular model of Parkinson's disease

机译：帕金森氏病细胞模型中酶活性的阻断抑制组织转谷氨酰胺酶介导的α-突触核蛋白的转酰胺作用

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Transamidation of a-synuclein by the Ca~(2+)-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 Ca~(2+) ionophore (A23187) induced activation of cellular tTG (measured by incorporation of the tTG-specific amine substrate 5-(biotinamido)penty-lamine (BAP) into cellular proteins) in neuroblastoma SH-SY5Y cells, only Z006 (0.3-30 uJVI) retained the capacity to completely inhibit tTG activity. Under these conditions B003 (3-300 fjuM) only partially blocked tTG activity whereas KCC009 (3-100 (jtM) failed to affect tTG activity at any of the concentrations used. Z006 (30 (aM) also blocked the tTG mediated incorporation of BAP into a-synuclein monomers and SDS-resistant multimers in vitro and in a-synuclein overexpressing SHSY5Y cells exposed to A23187 or the PD mimetic 1 -methyl-4-phenylpyridine (MPP~+). Moreover, Z006 (30 |a,M) substantially reduced formation of SDS-resistant a-synuclein multimers in SH-SY5Y cells exposed to A23187 or MPP~+ in the absence of BAP. We conclude that a-synuclein is a cellular substrate for tTG under conditions mimicking PD and blockade of tTG activity counteracts a-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.
机译：Ca-（2+）依赖性酶组织转谷氨酰胺酶（tTG，EC 2.3.2.13）对α-突触核蛋白的转酰胺作用涉及帕金森氏病（PD）。因此，tTG可能提供干预PD的新治疗靶点。在这里，我们首先评估了三种最近开发的不可逆的tTG活性部位抑制剂（B003，Z006和KCC009）在体外和活细胞中抑制tTG活性的效力和功效。在体外，发现所有化合物都是tTG活性的完全抑制剂，显示出Z006> B003> KCC009的效价等级等级（由IC-50值定义）。 Ca〜（2+）离子载体（A23187）诱导神经母细胞瘤SH-SY5Y细胞中细胞tTG的活化（通过将tTG特异性胺底物5-（biotinamido）penty-lamine（BAP）掺入细胞蛋白来测量） Z006（0.3-30 uJVI）保留了完全抑制tTG活性的能力。在这些条件下，B003（3-300 fjuM）仅部分阻断了tTG的活性，而KCC009（3-100（jtM）在任何所使用的浓度下均未影响tTG的活性; Z006（30（aM））也阻断了tTG介导的BAP的掺入。在体外以及在过量表达α-突触核蛋白的SHSY5Y细胞中暴露于a-突触核蛋白单体和抗SDS的多聚体中，该SHSY5Y细胞暴露于A23187或PD模拟的1-甲基-4-苯基吡啶（MPP〜+）。此外，Z006（30 | a，M）在没有BAP的情况下，暴露于A23187或MPP〜+的SH-SY5Y细胞中SDS抗性a-突触核蛋白多聚体的形成显着减少，我们得出结论，a-突触核蛋白是在模拟PD和阻断tTG活性的条件下tTG的细胞底物。在体外和在活细胞中，α-突触核蛋白都能抑制α-突触核蛋白的转氨作用和聚集，此外，我们的细胞模型似乎可以很好地识别细胞内tTG的候选抑制剂。

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