in-silico and in-vitro analysis of IL36RN mutations reveals critical residues for the function of the interleukin-36 receptor complex

Generalized pustular psoriasis (GPP) is a potentially life-threatening skin disease, associated with IL36RN mutations. IL36RN encodes the interleukin (IL)-36 receptor antagonist (IL-36Ra), a protein that downregulates the activity of IL-36 cytokines by blocking their receptor (IL-36R). While GPP can be treated with IL-36R inhibitors, the structural underpinnings of the IL-36Ra/IL-36R interaction remain poorly understood. Here, we sought to address this question by systematically investigating the effects of IL36RN mutations. We experimentally characterized the effects of 30 IL36RN variants on protein stability. In parallel, we used a machine-learning tool (Rhapsody) to analyse the IL-36Ra three-dimensional structure and predict the impact of all possible amino acid substitutions. This integrated approach identified 21 amino acids that are essential to IL-36Ra stability. We next investigated the effects of IL36RN changes on IL-36Ra/IL-36R binding and IL-36R signalling. By combining in-vitro assays and machine-learning with a second programme (mCSM), we identified 13 amino acids that are critical to IL-36Ra/IL36R engagement. Finally, we experimentally validated three representative predictions, further confirming the reliability of Rhapsody and mCSM. These findings shed light on the structural determinants of IL-36Ra activity, with potential to facilitate the design of new IL-36 inhibitors and aid the interpretation of IL36RN variants in diagnostic settings

TAB2, TRAF6 and TAK1 are involved in NF-κB activation induced by the TNF-receptor, Edar and its adaptator Edaradd

International audienceActivation of the NF-kappaB pathway by the TNF-receptor Edar (Ectodysplasin receptor) and its downstream adaptator Edaradd (Edar-associated death domain) is essential for the development of hair follicles, teeth, exocrine glands and other ectodermal derivatives. Dysfunction of Edar signalling causes hypohidrotic/anhidrotic ectodermal dysplasia (ED), a disorder characterized by sparse hair, lack of sweat glands and malformation of teeth. The Edar signalling pathway stimulates NF-kappaB transcription factors via an activation of the IkappaB kinase (IKK) complex. To gain further insight into the mechanism of IKK activation by Edar and Edaradd, we performed a yeast two-hybrid screen and isolated TAB2 (TAK1-binding protein 2) as a binding partner of Edaradd. TAB2 is an adaptator protein that brigdes TRAF6 (TNF-receptor-associated factor 6) to TAK1 (TGFbeta-activated kinase 1), allowing TAK1 activation and subsequent IKK activation. Here, we show that endogenous and overexpressed TAB2, TRAF6 and TAK1 co-immunoprecipitated with Edaradd in 293 cells. Moreover, we show that dominant negative forms of TAB2, TRAF6 and TAK1 blocked the NF-kappaB activation induced by Edaradd. These results support the involvement of the TAB2/TRAF6/TAK1 signalling complex in the Edar signal transduction pathway and have important implications for our understanding of NF-kappaB activation and EDs in human

Genomic architecture at the Incontinentia Pigmenti locus favours de novo pathological alleles through different mechanisms

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PW02-012 - First clinical description of an infant with DITRA

International audienc

PW02-012 - First clinical description of an infant with DITRA

International audienc

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti

The data that support the findings of this study are available from the corresponding authors upon request.International audienceThe NF-kB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-kB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-kB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients