Serpin-1a and serpin-6 regulate the Toll pathway immune homeostasis by synergistically inhibiting the Spätzle-processing enzyme CLIP2 in silkworm, Bombyx mori

Abstract

The Toll receptor signaling pathway is an important innate immune response of insects to pathogen infection; its extracellular signal transduction involves serine protease cascade activation. However, excessive or constitutive activation of the Toll pathway can be detrimental. Hence, the balance between activation and inhibition of the extracellular protease cascade must be tightly regulated to achieve favorable outcomes. Previous studies have shown that serpins—serine protease inhibitors—negatively regulate insect innate immunity by inhibiting extracellular protease cascade signaling. Although the roles of serpins in insect innate immunity are well described, the physiological mechanisms underlying their synergistic effects remain poorly understand. Here, we characterize the molecular mechanism by which serpin-1a and serpin-6 synergistically maintain immune homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Through in vitro biochemical assays and in vivo bioassays, we demonstrate that clip-domain serine protease 2 (CLIP2), as the Toll cascade-activating terminal protease, is responsible for processing proSpätzle1 to induce the expression of antimicrobial peptides. Further biochemical and genetic analyses indicate that constitutively expressed serpin-1a and inducible serpin-6 synergistically target CLIP2 to maintain homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Taken together, this study provides new insights into the precise regulation of Toll cascade activation signals in insect innate immune responses and highlights the importance and complexity of insect immune homeostasis regulation.

Effect of acute exposure of Hg on physiological parameters and transcriptome expression in silkworms (Bombyx mori)

“…Serpins, a widely distributed family of serine protease (SP) inhibitors, primarily regulate the Toll pathway, promoting the synthesis of antimicrobial peptides (AMPs) and activating the prophenoloxidase (proPO) system by inhibiting cascades of serine proteinase (66). Negative regulators such as serpin-5, serpin-3, and serpin-15 control insect innate immunity by inhibiting clip proteinase cascades, which trigger immune responses, including the Toll pathway and melanization (67)(68)(69)(70). In this study, the up-regulation of these serpins in the midgut or fat body suggests their potential role in the innate immune response to Hg exposure.…”

In-silicodocking platform with serine protease inhibitor (SERPIN) structures identifies host cysteine protease targets with significance for SARS-CoV-2

“…For example, tPA was identified as a PAI-1 target through SDS-PAGE with zymography in plasma samples, searching for SERPIN-protease high molecular weight complexes 59 . More systematic approaches include Co-immunoprecipitation of SERPIN-protease complexes and subsequent partner identification by precipitating either the protease 60 or the SERPIN 61 . Another strategy is the co-expression of SERPIN variants and select proteases in bacteria, followed by assaying residual protease activity to identify inhibitory variants 62 .…”