Melkonian Arek V, Weng Nielson, Palanski Brad A, Khosla Chaitan
Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
School of Medicine, Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2019;1967:263-274. doi: 10.1007/978-1-4939-9187-7_16.
Transglutaminase 2 (TG2) is a ubiquitous mammalian enzyme that is implicated in a variety of physiological processes and human diseases. Normally, extracellular TG2 is catalytically dormant due to formation of an allosteric disulphide bond between Cys370 and 371 of the enzyme. In this protocol, we describe a method to reduce this disulphide bond in living mice and to monitor the resulting in vivo TG2 activity. Briefly, exogenous thioredoxin-1 protein (TRX) is prepared and administered as a specific, physiologically relevant reductant of the Cys370-371 disulphide along with the small molecule 5-biotinamidopentylamine (5-BP) as a TG2 activity probe. Tissue cryosections are then analyzed by immunohistochemistry to ascertain the extent of 5-BP incorporation, which serves as a record of the redox state of TG2 in vivo. This protocol focuses on the modulation and measurement of TG2 in the small intestine, but we encourage investigators to evaluate it in their organ(s) of interest.
转谷氨酰胺酶2（TG2）是一种普遍存在于哺乳动物体内的酶，与多种生理过程和人类疾病相关。正常情况下，由于该酶的半胱氨酸370（Cys370）和371之间形成了变构二硫键，细胞外TG2处于催化休眠状态。在本实验方案中，我们描述了一种在活体小鼠中还原这种二硫键并监测由此产生的体内TG2活性的方法。简而言之，制备外源性硫氧还蛋白-1（TRX）并将其作为Cys370-371二硫键的特异性、生理相关还原剂进行给药，同时将小分子5-生物素酰胺基戊胺（5-BP）作为TG2活性探针。然后通过免疫组织化学分析组织冷冻切片，以确定5-BP掺入的程度，这可作为体内TG2氧化还原状态的记录。本实验方案着重于小肠中TG2的调节和测量，但我们鼓励研究人员在其感兴趣的器官中对其进行评估。
