TurboID biotin-tagging mass spectrometry identifies specific caspase-11-associated proteins regulating non-canonical inflammasome activation

Abstract While it has been demonstrated that cytosolic LPS can directly activate caspase11, the cellular processes regulating the non-canonical inflammasome response remain poorly defined. Caspase11 and caspase1 show substantial structural similarity, however, unlike the activation of caspase1 by NLR inflammasomes, there are no sensor or adaptor proteins known to be involved in transmitting cytosolic LPS signal to caspase11. Also, while caspase11 has been shown to associate with LPS, it lacks a characteristic LPS binding domain as observed in many other LPS binding proteins such as MD2 and LBP. Thus, we hypothesized that other effectors may be required to facilitate cytosolic LPS recognition. Moreover, the pathway is likely to be tightly regulated as caspase11 activation leads to highly inflammatory cell death. To identify novel regulators of caspase11, we generated immortalized macrophages expressing a caspase11-TurboID-DHFR chimeric protein. The destabilizing domain was included to avoid cell death induced by caspase11 over-expression. We used a TurboID biotin-tagging MS assay to detect proteins in close proximity to caspase11 pre and post cytosolic LPS introduction. Importantly, the TurboID assay permits recognition of transient interactions, typically missed by traditional IP. To validate relevance of putative hits, we used siRNA knockdown in BMDM. We’ve identified novel regulators specific for cytosolic LPS triggering. Several proteins interact with caspase11 only in the resting state, suggesting negative regulation to prevent pyroptosis. Among the identified regulators are kinases and proteins with pyrin and LRR domains, both common NLR features. This work was supported by the Intramural Research Program of NIAID, NIH.