The chart in the lower right summarizes the interaction results

The chart in the lower right summarizes the interaction results. Immunoprecipitation assay demonstrating the interaction between cIAPs and GLMN. cIAPs and GLMN co\immunoprecipitated with IpaH7.8. Colocalization of endogenous GLMN (red) and cIAP2 (green) in BMDMs, visualized by confocal microscopy. of the AIM2 inflammasome by poly dA:dT. GLMN binds specifically to the RING domain of both cIAPs, which inhibits their self\ubiquitination activity. These findings suggest that GLMN is a negative regulator of cIAP\mediated inflammasome activation, and highlight a unique Shigella stratagem to kill macrophages, promoting severe inflammation. Legionellatrigger activation of caspase\1 via the NLRC4 inflammasome in infected macrophages 3, 5. NLRC4 recognizes the bacterial flagellin 6, 7, inner rod protein 8, 9, and needle protein 10 of the bacterial type III secretion system (T3SS). Following recognition of these bacterial pathogen\associated molecular patterns (PAMPs), NLRC4 is assembled into the inflammasome complex, which induces proteolytic maturation of caspase\1 and pro\inflammatory cytokines such as IL\1 and IL\18, resulting in a form of cell death called pyroptosis 3, 5. In recent studies, gasdermin D, a member of the gasdermin protein family, was identified as the key caspase\1 substrate leading to pyroptosis 11, 12, 13. Caspase\1 (or caspase\11) cleaves gasdermin D to separate its N\terminal pore\forming domain from its C\terminal repressor domain 11, 12. The oligomerized N\terminal domain of gasdermin D permeabilizes the plasma membrane by forming large pores in the membrane Rabbit polyclonal to ZMYND19 that consequently cause membrane rupture and destruction of the cell (i.e., pyroptosis) 13. stimulates NLR inflammasomes by delivering various effectors and T3SS components into host cells via the T3SS 14, 15, 16, 17. We previously reported that induces rapid macrophage pyroptotic cell death, a prerequisite for bacterial Amfenac Sodium Monohydrate egress from macrophages, by delivering the IpaH7.8 E3 ubiquitin ligase effector via the T3SS, thereby activating NLRP3 and NLRC4 inflammasomes and caspase\1 18. In that study, we identified glomulin (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase\dependent inflammasome activation. Indeed, upregulation or downregulation of GLMN levels leads to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induce GLMN puncta that Amfenac Sodium Monohydrate colocalize with the active form of caspase\1 18. Although the mechanism of how GLMN is involved in inflammasome activation during infection remains unknown, the study suggested possess some as yet uncharacterized mechanisms to manipulate inflammasome activity, via the Amfenac Sodium Monohydrate interaction of IpaH7.8 and GLMN 18. Importantly, GLMN was originally identified through its association with glomuvenous malformations 19, and later, it was identified as acting as an inhibitor of Cullin\RBX1 E3 Amfenac Sodium Monohydrate ligases 20, 21. Structural and biochemical analyses showed that GLMN can associate with RBX1\containing Cullin\RING E3 ligase (CRL) by targeting the RING domain and masking its E2\binding surface, thereby inhibiting CRL E3 ligase activity 20, 21. Moreover, there is increasing evidence that ubiquitination modification of inflammasome component proteins is important for the regulation of inflammasome activity 22, 23, 24, 25, 26, 27. Given the interplay between GLMN and inflammasomes, we hypothesized that GLMN could have targets involved in modulating NLR inflammasome activation, for example, by interacting with and inhibiting certain putative RING\E3 ligases. Results GLMN targets cIAP1 and cIAP2 We screened a subset of host proteins, previously identified proteins capable of interacting with IpaH7.8 by GST pulldown with GST\IpaH7.8\coated beads 18, in\gel digestion and LC\TOF/TOF\MS analyses, for the ability to bind GLMN. Among the 18 proteins, we found cellular inhibitor of apoptosis protein (cIAP) 1 [also known as baculoviral IAP repeat\containing (BIRC) 2] bound GLMN in the yeast two\hybrid system using pPC86\GLMN and pDBleu\cIAP1 (positive control: pDBleu\IpaH7.8; negative control: pDBleu\empty; Fig?1A). cIAP1 belongs to the IAP family (also known as the BIRC family), whose members act as E3 ligases 28 and are critical regulators of multiple cellular pathways that control cell death, proliferation, and differentiation 28, 29. Because GLMN acts as.