一种使用化学交联检测炎性体诱导 ASC 寡聚化的简化方法。

A Streamlined Method for Detecting Inflammasome-Induced ASC Oligomerization Using Chemical Crosslinking.

机构信息

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

The Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.

出版信息

Methods Mol Biol. 2023;2691:155-164. doi: 10.1007/978-1-0716-3331-1_12.

DOI:10.1007/978-1-0716-3331-1_12

PMID:37355544

Abstract

The apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) functions as the integral adaptor protein between inflammasome sensors such as NOD-like receptor protein 3 (NLRP3) and the inflammatory caspase, caspase-1. Inflammasome sensor triggering allows recruitment of ASC and the formation of long amyloid-like ASC oligomers that enable binding and proximity-induced activation of caspase-1. The detection of ASC oligomerization thus constitutes a highly specific and direct test for inflammasome complex formation and activation. Here, we describe a simplified and streamlined method for the detection of ASC oligomers via Western blotting, using the chemical crosslinking reagent disuccinimidyl suberate.

摘要

凋亡相关斑点样蛋白包含一个半胱氨酸蛋白酶激活和募集结构域（ASC），作为衔接蛋白，连接模式识别受体如 NOD 样受体蛋白 3（NLRP3）和炎症半胱氨酸蛋白酶，半胱氨酸蛋白酶 1。炎症小体传感器的触发允许 ASC 的募集和长淀粉样 ASC 寡聚体的形成，从而使半胱氨酸蛋白酶 1 的结合和临近诱导激活。因此，ASC 寡聚体的检测构成了炎症小体复合物形成和激活的高度特异性和直接的检测方法。在这里，我们描述了一种通过 Western blot 检测 ASC 寡聚体的简化和优化方法，使用化学交联试剂二琥珀酰亚胺基琥珀酸酯。