Proteomics reveals altered lipid biosynthesis and keratin hyperphosphorylation in pachyonychia congenita

Pachyonychia congenita is a rare and painful skin disorder caused by dominant pathogenic variants in keratin genes (K6A/K6B/K6C/K16/K17), with no effective treatment. We developed a scalable, in-depth, and miniaturized mass spectrometry-based proteomics and phosphorylated proteomics analysis of full-thickness skin biopsies applied to 10 patients with pachyonychia congenita to elucidate pathogenic mechanisms and pinpoint therapeutic targets. We quantified 7200 protein groups on average from 2-mm snap-frozen skin samples, the most in-depth proteome coverage reported to date from a single-shot mass spectrometry analysis. Among the identified protein groups, ∼1400 proteins were differentially abundant in lesional versus nonlesional samples. Enrichment analysis points toward impaired mitochondrial function, hyperkeratinization, enhanced immune response, and a significant increase in cholesterol biosynthesis. Phosphoproteomics revealed hyperphosphorylation of specific sites in pachyonychia congenita-related keratins; confirmed the hyperactivation of EGFR and downstream kinases, including protein kinase C and Src, and disclosed p38 MAPK activation. All of these kinases have been reported to phosphorylate keratins. Our study expands upon current understanding of the consequences of EGFR pathway activation, including increased cholesterol biosynthesis, thereby renewing interest in the use of statins for pachyonychia congenita. Above all, it provides a solid foundation for the continued exploration of EGFR inhibitors and offers therapeutic avenues, particularly those using multikinase inhibitors.