Palanski Brad A, Khosla Chaitan
Biochemistry. 2018 Jun 19;57(24):3359-3363. doi: 10.1021/acs.biochem.8b00204. Epub 2018 Mar 28.
The catalytic activity of transglutaminase 2 (TG2), a ubiquitously expressed mammalian enzyme, is regulated by multiple post-translational mechanisms. Because elevated activity of TG2 in the extracellular matrix is associated with organ-specific diseases such as celiac disease and renal fibrosis, there is growing therapeutic interest in inhibitors of this enzyme. Cystamine, a symmetric disulfide compound, is one of the earliest reported TG2 inhibitors. Despite its widespread use as a tool compound to block TG2 activity in vitro and in vivo, its mechanism of action has remained unclear. Here, we demonstrate that cystamine irreversibly inhibits human TG2 ( k/ K = 1.2 mM min) via a mechanism fundamentally distinct from those proposed previously. Through mass spectrometric disulfide mapping and site-directed mutagenesis, we show that cystamine promotes the formation of a physiologically relevant disulfide bond between Cys370 and Cys371 that allosterically abrogates the catalytic activity of human TG2. This discovery led us to evaluate clinically useful thiol → disulfide oxidants for TG2 inhibitory activity. It is demonstrated that disulfiram, a relatively safe oral thiuram disulfide, is a fairly potent TG2 inhibitor ( k/ K = 8.3 mM min) and may therefore provide a practical tool for clinically validating this emerging therapeutic target in intestinal disorders such as celiac disease.
转谷氨酰胺酶2（TG2）是一种在哺乳动物中普遍表达的酶，其催化活性受多种翻译后机制调控。由于细胞外基质中TG2活性升高与乳糜泻和肾纤维化等器官特异性疾病相关，因此对该酶抑制剂的治疗兴趣日益浓厚。胱胺是一种对称二硫化物化合物，是最早报道的TG2抑制剂之一。尽管它作为一种工具化合物在体外和体内广泛用于阻断TG2活性，但其作用机制仍不清楚。在此，我们证明胱胺通过一种与先前提出的机制根本不同的机制不可逆地抑制人TG2（k/K = 1.2 mM·min）。通过质谱二硫键图谱分析和定点诱变，我们表明胱胺促进Cys370和Cys371之间形成生理相关的二硫键，从而变构消除人TG2的催化活性。这一发现促使我们评估具有临床应用价值的硫醇→二硫化物氧化剂的TG2抑制活性。结果表明，双硫仑是一种相对安全的口服秋兰姆二硫化物，是一种相当有效的TG2抑制剂（k/K = 8.3 mM·min），因此可能为在乳糜泻等肠道疾病中临床验证这一新兴治疗靶点提供一种实用工具。
