A conformation-locking inhibitor of SLC15A4 with TASL proteostatic anti-inflammatory activity.

Dysregulation of pathogen-recognition pathways of the innate immune system is associated with multiple autoimmune disorders. Due to the intricacies of the molecular network involved, the identification of pathway- and disease-specific therapeutics has been challenging. Using a phenotypic assay monitoring the degradation of the immune adapter TASL, we identify feeblin, a chemical entity which inhibits the nucleic acid-sensing TLR7/8 pathway activating IRF5 by disrupting the SLC15A4-TASL adapter module. A high-resolution cryo-EM structure of feeblin with SLC15A4 reveals that the inhibitor binds a lysosomal outward-open conformation incompatible with TASL binding on the cytoplasmic side, leading to degradation of TASL. This mechanism of action exploits a conformational switch and converts a target-binding event into proteostatic regulation of the effector protein TASL, interrupting the TLR7/8-IRF5 signaling pathway and preventing downstream proinflammatory responses. Considering that all components involved have been genetically associated with systemic lupus erythematosus and that feeblin blocks responses in disease-relevant human immune cells from patients, the study represents a proof-of-concept for the development of therapeutics against this disease.

Structural basis for recruitment of TASL by SLC15A4 in human endolysosomal TLR signaling.

Toll-like receptors (TLRs) are a class of proteins that play critical roles in recognizing pathogens and initiating innate immune responses. TASL, a recently identified innate immune adaptor protein for endolysosomal TLR7/8/9 signaling, is recruited by the lysosomal proton-coupled amino-acid transporter SLC15A4, and then activates IRF5, which in turn triggers the transcription of type I interferons and cytokines. Here, we report three cryo-electron microscopy (cryo-EM) structures of human SLC15A4 in the apo monomeric and dimeric state and as a TASL-bound complex. The apo forms are in an outward-facing conformation, with the dimeric form showing an extensive interface involving four cholesterol molecules. The structure of the TASL-bound complex reveals an unprecedented interaction mode with solute carriers. During the recruitment of TASL, SLC15A4 undergoes a conformational change from an outward-facing, lysosomal lumen-exposed state to an inward-facing state to form a binding pocket, allowing the N-terminal helix of TASL to be inserted into. Our findings provide insights into the molecular basis of regulatory switch involving a human solute carrier and offers an important framework for structure-guided drug discovery targeting SLC15A4-TASL-related human autoimmune diseases.

SLC15A4 controls endolysosomal TLR7-9 responses by recruiting the innate immune adaptor TASL.

Endolysosomal Toll-like receptors (TLRs) play crucial roles in immune responses to pathogens, while aberrant activation of these pathways is associated with autoimmune diseases, including systemic lupus erythematosus (SLE). The endolysosomal solute carrier family 15 member 4 (SLC15A4) is required for TLR7/8/9-induced responses and disease development in SLE models. SLC15A4 has been proposed to affect TLR7-9 activation through its transport activity, as well as by assembling an IRF5-activating complex with TASL, but the relative contribution of these functions remains unclear. Here, we show that the essential role of SLC15A4 is to recruit TASL to endolysosomes, while its transport activity is dispensable when TASL is tethered to this compartment. Endolysosomal-localized TASL rescues TLR7-9-induced IRF5 activation as well as interferon ß and cytokine production in SLC15A4-deficient cells. SLC15A4 acts as signaling scaffold, and this function is essential to control TLR7-9-mediated inflammatory responses. These findings support targeting the SLC15A4-TASL complex as a potential therapeutic strategy for SLE and related diseases.

NLRP3 leucine-rich repeats control induced and spontaneous inflammasome activation in cryopyrin-associated periodic syndrome.

BACKGROUND: The cryopyrin-associated periodic syndromes (CAPS) comprise a group of rare autoinflammatory diseases caused by gain-of-function mutations in the NLRP3 gene. NLRP3 contains a leucine-rich repeats (LRR) domain with a highly conserved exonic organization that is subjected to extensive alternative splicing. Aberrant NLRP3 inflammasome assembly in CAPS causes chronic inflammation; however, the mechanisms regulating inflammasome function remain unclear. OBJECTIVE: We aimed to elucidate the mechanisms regulating NLRP3-mediated autoinflammation in human disease, characterizing the role of LRR in inflammasome activation. METHODS: We analyzed sequence read archive data to characterize the pattern of NLRP3 splicing in human monocytes and investigated the role of each LRR-coding exon in inflammasome regulation in genetically modified U937 cells representing CAPS and healthy conditions. RESULTS: We detected a range of NLRP3 splice variants in human primary cells and monocytic cell lines, including 2 yet-undescribed splice variants. We observe that lipopolysaccharides affect the abundance of certain splice variants, suggesting that they may regulate NLRP3 activation by affecting alternative splicing. We showed that exons 4, 5, 7, and 9 are essential for inflammasome function, both in the context of wild-type NLRP3 activation by the agonist molecule nigericin and in a model of CAPS-mediated NLRP3 inflammasome assembly. Moreover, the SGT1-NLRP3 interaction is decreased in nonfunctional variants, suggesting that alternative splicing may regulate the recruitment of proteins that facilitate inflammasome assembly. CONCLUSION: These findings demonstrate the contribution of the LRR domain in inflammasome function and suggest that navigating LRR exon usage within NLRP3 is sufficient to dampen inflammasome assembly in CAPS.