Innate immune signaling pathways comprise multiple proteins that promote inflammation. This multistep means of information transfer suggests that complexity is a prerequisite for pathway design. Here, we test this hypothesis by studying caspases that regulate inflammasome-dependent inflammation. Several caspases differ in their ability to recognize bacterial lipopolysaccharide (LPS) and cleave interleukin-1β (IL-1β). No caspase is known to contain both activities, yet distinct caspases with complementary activities bookend an LPS-induced pathway to IL-1β cleavage. Using caspase-1/4 hybrid proteins present in canines as a guide, we identified molecular determinants of IL-1β cleavage specificity within human and murine caspase-1. This knowledge enabled the redesign of human caspase-4 to operate as a one-protein signaling pathway, which intrinsically links LPS detection to IL-1β cleavage and release, independent of inflammasomes. We identified caspase-4 homologs in multiple carnivorans that display the activities of redesigned human caspase-4. These findings illustrate natural signaling pathway diversity and highlight how multistep innate immune pathways can be condensed into a single protein.

先天免疫信号通路包含多种促进炎症的蛋白质。这种多步骤的信息传递方式表明，复杂性是通路设计的先决条件。在这里，我们通过研究调节炎症小体依赖性炎症的半胱天冬酶来验证这一假设。几种半胱天冬酶识别细菌脂多糖 (LPS) 和切割白细胞介素 1β (IL-1β) 的能力不同。已知没有 caspase 包含这两种活性，但具有互补活性的不同 caspase 终止了 LPS 诱导的 IL-1β 切割途径。以犬科动物中存在的 caspase-1/4 杂合蛋白为指导，我们确定了人和鼠 caspase-1 中 IL-1β 切割特异性的分子决定因素。这一知识使人类 caspase-4 的重新设计能够作为单蛋白信号通路发挥作用，这在本质上将 LPS 检测与 IL-1β 裂解和释放联系起来，与炎症小体无关。我们在多种食肉动物中鉴定出 caspase-4 同源物，这些同源物显示出重新设计的人类 caspase-4 的活性。这些发现说明了自然信号通路的多样性，并突出了多步先天免疫通路如何浓缩成单一蛋白质。