O02 Ubiquitomics of CYLD-deficient skin tumours reveals dysregulation of hair follicle keratinocyte processes

Abstract Introduction and aims CYLD cutaneous syndrome (CCS) is an inherited hair follicle tumour syndrome where biallelic loss of function of the deubiquitinase enzyme CYLD gives rise to cylindroma skin tumours. The role of CYLD in skin biology is not well characterized but it is known to regulate several cell signalling pathways through deubiquitination of effector proteins. We aimed to determine how loss of CYLD function can lead to CCS skin tumour formation by studying the ubiquitome of CCS tumours and healthy skin. Methods Healthy skin (n = 5) and CCS tumour tissue (n = 5) were collected from flash frozen samples. Lysed homogenates were trypsin-digested and the resulting peptides underwent diglycine immunopurification for subsequent liquid chromatography–tandem mass spectrometry analysis on a Bruker Tims-TOF HT (Billerica, MA, USA) mass spectrometer. We utilized a data-independent acquisition method adapted for diglycine-containing peptides to generate mass spectrometry data. Peptide identification was performed by searching mass spectrometry data against an in silico neural network derived spectral library with diglycine modification. Results We identified 1273 ubiquitination sites across 649 proteins in CCS tumour and control samples. After performing t-tests (P < 0.05), we found 305 ubiquitination sites that were either unique to their respective condition or were differentially regulated. Analysis of ubiquitinated peptide sequences uncovered cell surface receptors with increased ubiquitination in CCS tumour samples including the insulin receptor, which had ubiquitination modifications at its kinase domain, suggesting increased insulin signalling in CCS tumours. Gene set enrichment analysis of CCS tumour ubiquitinated substrates revealed increased ubiquitination of secretory granules and extracellular exosomes, suggesting that loss of CYLD may disrupt regulation of protein secretion processes. Conclusions Our findings describe the ubiquitome of CCS keratinocytes, providing insights into how loss of CYLD function can affect biological processes in skin.