Proteasome-mediated degradation of keratins 7, 8, 17 and 18 by mutant KLHL24 in a foetal keratinocyte model: Novel insight in congenital skin defects and fragility of epidermolysis bullosa simplex with cardiomyopathy

Abstract

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (ΔN28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in ΔN28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in ΔN28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients’ keratinocyte clonogenicity impairment.

Towards a Better Understanding of Genotype–Phenotype Correlations and Therapeutic Targets for Cardiocutaneous Genes: The Importance of Functional Studies above Prediction

“…This result was in line with a reduced expression and phenotype of the explanted heart of one of the patients [101]. Meanwhile, another study reported that KLHL24 is also responsible for the turnover of foetal-like keratins 7,8,17 and 18 [103], while degradation of keratin 14 may be higher in foetal-like hiPSC-derived keratinocytes compared to adult keratinocytes [102]. These data would explain why patients develop congenital aplasia cutis but only mild skin fragility later in life.…”

“…For instance, KLHL24 protein activity, whether through loss or gain of function, causes a strong dose-dependent phenotype in the heart, which seems directly related to expression of its target desmin [101,107]. The relationship to keratins in the skin is less straightforward and requires more cohesive data that may now be emerging with new studies [102][103][104]. Functional studies combined also suggest that the phenotype associated with DP reduction is both dose-and organ-dependent in nature, which is important knowledge for future interventions.…”

In silico analysis of the profilaggrin sequence indicates alterations in the stability, degradation route, and intracellular protein fate in filaggrin null mutation carriers

“…Our analysis indicated that the monomers generated by SASPase cleavage are relatively stable, given their minor decrease in the N-terminal PSI, but a significant increase in the C-terminal PSI and a drop in GHI value; this likely permits their long-lasting functionality required for cross-linking of the proteins within the cornified envelope, and aggregation and collapse of the IF-based cytoskeleton. Intriguingly, the UPS system has been previously implicated in the degradation and turnover of keratins ( Yamazaki et al, 2012 ); increased keratin turnover is observed in a subtype of epidermolysis bullosa simplex (EBS) patients, resulting in shortened lifespan, replicative senescence, and decreased cellular resistance in keratinocytes ( Logli et al, 2022 ).…”

In silico analysis of the profilaggrin sequence indicates alterations in the stability, degradation route, and intracellular protein fate in filaggrin null mutation carriers

“…Our analysis indicated that the monomers generated by SASPase cleavage are relatively stable, given their minor decrease in the Nterminal PSI, but a signi cant increase in the C-terminal PSI and a drop in GHI value; this likely permits their long-lasting functionality required for cross-linking of the proteins within the corni ed envelope, and aggregation and collapse of the IF-based cytoskeleton. Intriguingly, the UPS system has been previously implicated in the degradation and turnover of keratins 182 ; increased keratin turnover is observed in a subtype of epidermolysis bullosa simplex (EBS) patients, resulting in shortened lifespan, replicative senescence, and decreased cellular resistance in keratinocytes 183 .…”