TY - JOUR
T1 - in-silico and in-vitro analysis of IL36RN mutations reveals critical residues for the function of the interleukin-36 receptor complex
AU - Hassi, Niina K
AU - Weston, Timir
AU - Rinaldi, Giulia
AU - Ng, Joseph C
AU - Smahi, Asma
AU - Twelves, Sophie
AU - Davan-Wetton, Camilla
AU - Fakhreddine, Dana
AU - Fraternali, Franca
AU - Capon, Francesca
N1 - Funding Information:
The authors are very grateful to Claire Peet and Luc Francis for their assistance. This research was supported by the National Institute for Health and Care Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas’ NHS Foundation Trust and King's College London (guysbrc-2012-1). The authors also received funding from the Medical Research Council, the Biotechnology and Biological Sciences Research Council (BBSRC) (grants MR/L01257X/2 and BB/T002212/1 to FF) and the British Skin Foundation (grant 008S/22 to FC and FF). ST was supported by the King's Bioscience Institute and the Guy's and St Thomas' Charity Prize PhD Programme in Biomedical and Translational Science. CDW and DF were funded by the BBSRC through the London Interdisciplinary Doctoral Training Partnership (LIDo-DTP) [grants BB/M009513/1 and BB/T008709/1]. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. None of the funders were involved in the study design, data collection, data analysis, or manuscript preparation. Conceptualization: FC, FF; Formal Analysis: GR, JCN, TW; Funding Acquisition: FC, FF; Investigation: CD-W, DF, NKH, ST; Resources: AS; Supervision: FC, FF; Writing-Review and Editing: FC, FF, GR, JCN, TW; Writing-Original Draft Preparation: FC.
Funding Information:
The authors are very grateful to Claire Peet and Luc Francis for their assistance. This research was supported by the National Institute for Health and Care Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London (guysbrc-2012-1). The authors also received funding from the Medical Research Council, the Biotechnology and Biological Sciences Research Council (BBSRC) (grants MR/L01257X/2 and BB/T002212/1 to FF) and the British Skin Foundation (grant 008S/22 to FC and FF). ST was supported by the King's Bioscience Institute and the Guy's and St Thomas' Charity Prize PhD Programme in Biomedical and Translational Science. CDW and DF were funded by the BBSRC through the London Interdisciplinary Doctoral Training Partnership (LIDo-DTP) [grants BB/M009513/1 and BB/T008709/1].
Publisher Copyright:
© 2023
PY - 2023/12
Y1 - 2023/12
N2 - Generalized pustular psoriasis is a potentially life-threatening skin disease, associated with IL36RN disease alleles. IL36RN encodes the IL-36 receptor antagonist (IL-36Ra), a protein that downregulates the activity of IL-36 cytokines by blocking their receptor (IL-36R). Although generalized pustular psoriasis can be treated with IL-36R inhibitors, the structural underpinnings of the IL-36Ra/IL-36R interaction remain poorly understood. In this study, we sought to address this question by systematically investigating the effects of IL36RN sequence changes. We experimentally characterized the effects of 30 IL36RN variants on protein stability. In parallel, we used a machinelearning tool (Rhapsody) to analyze 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 for IL-36Ra stability. We next investigated the effects of IL36RN changes on IL-36Ra/IL-36R binding and IL-36R signaling. Combining invitro assays and machine learning with a second program (mCSM), we identified 13 amino acids that are critical for 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.
AB - Generalized pustular psoriasis is a potentially life-threatening skin disease, associated with IL36RN disease alleles. IL36RN encodes the IL-36 receptor antagonist (IL-36Ra), a protein that downregulates the activity of IL-36 cytokines by blocking their receptor (IL-36R). Although generalized pustular psoriasis can be treated with IL-36R inhibitors, the structural underpinnings of the IL-36Ra/IL-36R interaction remain poorly understood. In this study, we sought to address this question by systematically investigating the effects of IL36RN sequence changes. We experimentally characterized the effects of 30 IL36RN variants on protein stability. In parallel, we used a machinelearning tool (Rhapsody) to analyze 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 for IL-36Ra stability. We next investigated the effects of IL36RN changes on IL-36Ra/IL-36R binding and IL-36R signaling. Combining invitro assays and machine learning with a second program (mCSM), we identified 13 amino acids that are critical for 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.
UR - http://www.scopus.com/inward/record.url?scp=85172307689&partnerID=8YFLogxK
U2 - 10.1016/j.jid.2023.06.191
DO - 10.1016/j.jid.2023.06.191
M3 - Article
C2 - 37414245
SN - 0022-202X
VL - 143
SP - 2468-2475.e6
JO - Journal of Investigative Dermatology
JF - Journal of Investigative Dermatology
IS - 12
ER -