Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia.
EN-FIST Centre of Excellence, Ljubljana, Slovenia.
Methods Mol Biol. 2023;2696:257-267. doi: 10.1007/978-1-0716-3350-2_17.
NACHT-, LRR-, and PYD-containing protein 3 (NLRP3) is a member of AAA+ ATPase family that upon activation forms inflammasomes. Several studies demonstrated that ATP binding and hydrolysis are important for NLRP3 function as an inflammasome sensor. Furthermore, compounds targeting ATP binding motifs and interfering with ATPase activity of NLRP3 inhibit NLRP3 inflammasome formation. Measuring ATPase activity of proteins and binding of radiolabeled ATP to specified proteins are well-established methods that require purified protein. Here, we describe a method for assessing NLRP3 binding to ATP using ATP-conjugated beads and lysates of cells that either express endogenous NLRP3 or are transfected with plasmids encoding NLRP3. Efficiency of binding is followed after elution from the beads and detection with Western blot and immunolabelling. The method can be used to evaluate the functionality of NLRP3 variants or to check whether compounds or NLRP3 binding partners interfere with binding of ATP.
NACHT、LRR 和 PYD 结构域包含蛋白 3（NLRP3）是 AAA+ ATP 酶家族的一员，其在被激活后会形成炎性体。多项研究表明，ATP 结合和水解对于 NLRP3 作为炎性体传感器的功能很重要。此外，靶向 ATP 结合基序并干扰 NLRP3 ATP 酶活性的化合物可抑制 NLRP3 炎性体的形成。测量蛋白质的 ATP 酶活性以及放射性标记的 ATP 与特定蛋白质的结合是两种已建立的方法，这些方法都需要纯化的蛋白质。在这里，我们描述了一种使用 ATP 偶联珠和表达内源性 NLRP3 的细胞裂解物或转染编码 NLRP3 的质粒的细胞裂解物来评估 NLRP3 与 ATP 结合的方法。在用 Western blot 和免疫标记检测洗脱后的珠后，可检测到结合效率。该方法可用于评估 NLRP3 变体的功能，或检查化合物或 NLRP3 结合伴侣是否干扰 ATP 的结合。
