Thermal proteome profiling reveals meltome upon NLRP3 inflammasome activation

NLR family pyrin domain containing 3 (NLRP3) involves in inflammasome complex assembly and innate immunity. Activation of the NLRP3 inflammasome induces conformational alterations in protein complexes, influencing their interactions with other molecules, which in turn affects protein thermal stability. To investigate the proteome-wide thermal stability alterations induced by NLRP3 inflammasome activation, we conducted a comprehensive analysis of meltome dynamics using thermal proteome profiling (TPP). Our analysis identified 337 proteins exhibiting alterations in thermal stability upon NLRP3 inflammasome activation. Subsequently, we validated three proteins by the cellular thermal shift assay (CETSA). Notably, our findings reveal that the majority of these proteins tend to cluster into distinct macromolecular complexes. Furthermore, we identify FAM120A as a novel NLRP3 binding partner, with its suppression enhancing caspase-1 activation and IL-1β release in response to NLRP3 agonist. Collectively, these data provide a comprehensive framework for understanding the mechanisms of NLRP3 inflammasome activation and underscore the utility of TPP in exploring proteome-wide thermal stability changes during signaling transduction.含pyrin结构域的NLR家族蛋白3（NLRP3）参与炎症小体复合物组装及先天免疫过程。NLRP3炎症小体的激活会诱导蛋白质复合物发生构象改变，影响其与其他分子的相互作用，进而改变蛋白质的热稳定性。为探究NLRP3炎症小体激活引发的蛋白质组范围热稳定性变化，我们利用热蛋白质组分析（TPP）对熔解组动态展开全面分析。结果显示，NLRP3炎症小体激活后，共有337种蛋白质的热稳定性发生改变。随后，我们通过细胞热位移分析（CETSA）验证了其中三种蛋白质。值得注意的是，研究发现这些蛋白质大多倾向于聚集形成不同的大分子复合物。此外，我们鉴定出FAM120A是NLRP3的新型结合伙伴，抑制FAM120A可增强NLRP3激动剂诱导的caspase-1激活及IL-1β释放。综上，这些数据为理解NLRP3炎症小体激活机制提供了全面框架，并凸显了TPP在探索信号转导过程中蛋白质组范围热稳定性变化的应用价值。