Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Toxicol Appl Pharmacol. 2012 Sep 15;263(3):273-80. doi: 10.1016/j.taap.2012.06.019. Epub 2012 Jul 4.
Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs(III)) and its intermediate metabolites such as monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA(III) and DMA(III)) but not by iAs(III). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA(III) directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation.
砷与生物分子的结合被认为是主要的毒性机制之一，这也可能与砷对人类的致癌风险有关。同时，砷也已知能激活磷酸化依赖的信号通路，包括表皮生长因子受体、丝裂原活化蛋白激酶和胰岛素/胰岛素样生长因子-1 通路。这些信号通路起源于受体酪氨酸激酶水平，其磷酸化状态受蛋白酪氨酸磷酸酶（PTP）活性的调节。可逆的酪氨酸磷酸化受蛋白酪氨酸激酶和磷酸酶的平衡作用控制，调节着参与控制细胞增殖、黏附和迁移的重要信号通路。在本研究中，我们专注于细胞 PTP 与毒性三价亚砷酸盐（iAs(III)）及其中间代谢物（如一甲基砷酸（MMA(III)）和二甲基砷酸（DMA(III)））在体外的相互作用，然后使用重组 PTP（如 PTP1B 和 CD45）确定 PTP 中的砷结合位点。有趣的是，观察到 PTP1B（细胞质形式）或 CD45（受体连接形式）的活性被两种甲基化代谢物（即 MMA(III)和 DMA(III)）强烈抑制，但不受 iAs(III)的影响。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）清楚地证实，有机中间产物 DMA(III)直接与 PTP1B 的活性位点半胱氨酸残基（如 Cys215）结合，导致酶活性抑制。这些结果表明，砷暴露可能通过 PTP 失活扰乱细胞信号通路。
