Protein degradation is a highly coordinated process with multiple levels of regulation, including both targeted and autodegradation. This sophisticated cascade of protein turnover must be precisely balanced to maintain proper physiological function. A recent article published in Nature Genetics reports the discovery of gene with protein-truncating mutations that lead to the skin condition epidermolysis bullosa, which is characterized by tendency to blister, itching and other abnormalities. The authors found 5 patients all with start codon mutations in the KLHL24 gene, which encodes Kelch-like protein 24, a substrate receptor of the cullin 3 (CUL3)–RBX1–KLHL24 ubiquitin ligase complex.
The mutant proteins from these patients were found to be stabilized, with increased levels in patient samples, leading the authors to hypothesize that KLHL24 may target a substrate that is important for the structural integrity of the skin. Indeed, through mass spectrometry and biochemical analysis, they identify keratin 14 (KRT14) as a KLHL24 substrate, and find that KRT14 levels are decreased in patient samples. Keratin 14 is an intermediate filament component important for maintaining keratinocyte integrity and mutations in the gene are found in some epidermolysis bullosa patients. The authors further show that KLHL24 is autoubiquitinated and that the truncated mutant has reduced levels of autoubiquitination, stabilizing the protein. This increased KLHL24 stability leads to increased KRT14 degradation, resulting in the skin fragility phenotype observed in the patients.
Although dynamic regulation of keratins by the ubiquitin–proteasome system had been proposed, no targeting E3 ligases had been identified. This work established KLHL24 as a keratin-targeting E3 ligase.
We spoke with authors Dr. Xu Tan and Dr. Yong Yang to get some background on their research.
Can you briefly describe how you found the KLHL24 mutations in these different patients?
The first three epidermolysis bullosa (EB) patients were first screened for the 18 previously known causative genes but no mutations were found. Then we performed whole exome sequencing and pinned down only one common variant gene among all three patients, namely KLHL24. We then acquired samples from two additional patients without mutations in the 18 known causative genes and used Sanger sequencing to show that both of them also have the mutations in the same KLHL24 gene, confirming that this is a new causative gene of EB.
All the patients you studied had start codon mutations leading to truncations in the protein. This must have been intriguing. What where your initial thoughts about this finding?
We were shocked. The first thought was that these must be gain-of-function mutations, unlike all the other EB mutations, which are loss-of-function mutations that can occur all over the places.
You very nicely demonstrate a model whereby Keratin 14 is an ubiquitination substrate of KLHL24, and that the truncated mutant is stabilized, thus leading to greater Keratin 14 degradation and the skin fragility phenotype. Can you walk us through how you teased apart this model? What do you consider the key piece of evidence that supports this model?
We used an unbiased “pull down + mass spectrometry” method to look for the binding proteins to the substrate binding domain of KLHL24 and Keratin 14 was the only one we found that specifically binds KLHL24 but not a carefully designed mutant that is predicted structurally to lose the substrate binding capacity. We immediately verified the binding and also showed that knocking down/overexpressing KLHL24 can increase/decrease Keratin 14 levels. A key piece of evidence is that transfection of KLHL24 in cell lines can boost Keratin 14 ubiquitination. Afterwards, we obtained two important pieces of in vivo evidence to show the anti-correlation of KLHL24 level and Keratin 14 level (in human skin samples and a knock-in mouse model), nicely confirming that Keratin 14 is a ubiquitination substrate of KLHL24.
You make a knock-in mouse, which recapitulates the decreased Keratin 14 levels similar to what is seen in patients, but not the skin fragility phenotype. Can you comment on why this might be so?
Many differences exist between human and mouse skin, the most obvious is the presence of fur in the mouse skin, which might afford better mechanic support of the epidermis than that in the human skin. In addition, there is actually a small but significant difference between the degrees of Keratin 14 decrease in patients and the mouse model (~70% decrease in patients vs. ~50% decrease in mice). Previously mouse models having ~50% decrease of Keratin 14 (the Krt14+/- mouse model) also did not show skin fragility. We don’t yet know the reason for the differential decrease of levels in human and mouse skin but are working on finding out the answers.
Do your findings have any potential implications for novel therapies for epidermolysis bullosa?
Absolutely, as I mentioned these are the first gain-of-function mutations found for EB, which should be easier to target therapeutically than loss-of-function mutations. Inhibiting KLHL24 in patients that we identified with these types of mutations should be able to effectively treat the conditions. We are now actively working on finding a specific KLHL24 inhibitor. In addition, because KLHL24 is a negative regulator of Keratin 14, other EB patients with partial loss-of-function mutations of Keratin 14 could also be helped by treatment with a KLHL24 inhibitor. In general, drug development targeting the ubiquitin-proteasome pathway has been given high hopes but it is not very obvious how to target the pathway specifically. Our studies provide a good example showing the importance of autoubiquitination of an E3 ligase, which might suggest previously over-looked strategies to target E3s.