Mapping the proteolytic landscape in LEKTI deficiency human skin equivalent to model Netherton syndrome

Rare diseases present a group of heterogeneous conditions, affecting 1 in 200.000 newborns, without effective treatments. Netherton syndrome (NS) is a rare and debilitating skin disease that is characterised by severe skin barrier disruption and secondary skin inflammation. NS is caused by loss-of-function mutations in the serine protease inhibitor kazal-type 5 (SPINK5) gene, which results in the defective function of the corresponding gene product called lympho-epithelial kazal-type related inhibitor (LEKTI). So far, a limited number of proteases, including kallikrein (KLK 5) and KLK7, have been proposed as therapeutic targets for treatment. However, most of these targets have been proposed based on murine disease models. Furthermore, limited data exists of the overall biological landscape of NS in terms of protein changes. As NS pathogenesis is driven by uncontrolled protease activity, it is imperative to investigate the overall proteolytic landscape to thoroughly understand how the intricate epidermal protease network underlies the disease. This is especially important to identify novel protease targets and to evaluate efficacy of therapeutic interventions.
Therefore, the main objective of this study was to establish a disease model that could substitute NS patient skin or animal models. This model can be used for extensive protein and substrate characterisation, which could identify molecules of therapeutic interest. Firstly, I describe how to identify a compatible cell model by comparing three types of keratinocytes to establish a human skin equivalent. Primary neonatal keratinocytes were successfully used to generate a human skin equivalent (HSE) model where Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated SPINK5 knockout keratinocytes were used to generate a corresponding NS-like disease model. Then, I utilised the established models to perform an extensive characterisation of protein and substrate landscape by global proteome, N-terminomics, and targeted proteomics. The obtained biological landscape determined that LEKTI deficiency conferred few changes on the protein level. However, the substrate characterisation highlighted an increased degradation of epidermal proteins related to skin differentiation. Furthermore, targeted proteomics identified cathpesin V (CTSV) as significantly decreased. Finally, I used proteomics to evaluate the phenotypic changes of a LEKTI-deficient HSE upon protein depletion of desquamation proteases, including KLK5, KLK7, and CTSV, mediated by small interfering RNA (siRNA). The results showed partial rescue when KLK5 and KLK7 protein were depleted. However, CTSV protein depletion showed increased skin barrier impairment. In conclusion, the use of advanced tissue engineering to model rare diseases in combination with proteomics methods can provide insights into the biological landscape of NS and shows potential for target exploration and drug evaluation.