Comparative ubiquitinomics of human skin reveals insulin receptor ubiquitination as a regulator of collagen secretion

Abstract Ubiquitination is central to skin homeostasis and disease, however the cutaneous ubiquitinome remains poorly characterised. We investigated spatial patterns of ubiquitination in human skin, discovering specific ubiquitin linkages located within the hair follicle and epidermis. To create a comprehensive physiological profile of the ubiquitinated proteins within the skin, we profiled healthy skin and CYLD cutaneous syndrome (CCS) skin tumours through di-glycine remnant immunopurification proteomics, enabling identification of 1605 ubiquitin sites across 731 proteins. The healthy cutaneous ubiquitinome comprised substrate classes including keratins, S100 proteins and histones. Predictive upstream enzyme analysis ranked the most frequent E3 ligase as NEDD4, and the most frequent deubiquitinases included USP7 and CYLD. Analysis of the CCS tumor ubiquitinome, characterized by catalytically inactivated CYLD somatic mutations and prominent extracellular matrix (ECM) secretion, identified 251 differentially ubiquitinated sites compared to healthy skin. The CCS ubiquitinome was enriched for cell division and differentiation proteins, including predicted CYLD substrate insulin receptor (INSR). Secretome analysis of INSR knockdown CCS primary cells revealed reduced secretion of basement membrane proteins, particularly COL7A1. By defining how ubiquitination modulates pathophysiological ECM secretory output, our study highlights the ubiquitin post-translational system as a broader determinant of tissue architecture. Competing Interest Statement The authors have declared no competing interest. Footnotes Conflict of interest statement: “All authors declare no relevant conflicts of interest.”