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

Abstract Ubiquitination is central to skin homeostasis, however the cutaneous ubiquitinome remains poorly characterised. We investigated ubiquitination architecture in human skin, discovering ubiquitin linkage type localisation within the hair follicle and epidermis. We profiled healthy skin through di-glycine remnant immunopurification proteomics, identifying 1465 sites across histone, S100 and keratin protein classes. Predictive upstream enzyme analysis ranked the most frequent E3 ligases and deubiquitinases as NEDD4, USP7 and CYLD. Analysis of CCS tumours, characterised 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 secretion, our study highlights the ubiquitin system as a broader determinant of tissue architecture.