Targeted Proteolysis of Plectin Isoform 1a Accounts for Hemidesmosome Dysfunction in Mice Mimicking the Dominant Skin Blistering Disease EBS-Ogna

Abstract

Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.

The cytolinker plectin regulates nuclear mechanotransduction in keratinocytes

“…In future studies, investigation of how other types of plectin mutations, such as in EBS Ogna (Walko et al, 2011), and EBS caused by keratin mutations affect nuclear morphology and gene expression will be of great importance. Recent mRNA expression profiling studies, implicating pathways related to interleukins, lipid metabolism and keratinisation, suggest that transcriptional mechanisms might indeed play a role in EBS (Bchetnia et al, 2012;Lu et al, 2007).…”

“…Samples were mounted on glass slides with Mowiol reagent. Primary antibodies were against the following: lamin A/C (Santa Cruz Biotechnology, Ms 636, 1:200), K14 (Cancer Research UK, Ms LL002, 1:500), K14 (Covance, Rb polyclonal, 1:5000), α6 integrin (BD Biosciences, Rat GoH3, 1:100), paxillin (BD Biosciences, Ms 177, 1:200), plectin (Ms 10F6, 1:2) (Walko et al, 2011) and nesprin-3 (gift from Arnoud Sonnenberg, Netherlands Cancer Institute, Amsterdam, The Netherlands; rabbit polyclonal, 1:100). F-actin was labelled with phalloidin-Alexa-Fluor-488 (Life Technologies, 1:500).…”

Calpain-Mediated Integrin Deregulation as a Novel Mode of Action for the Anticancer Gallium Compound KP46

“…Cells were plated in 6-well dishes at a density of 1 Â 10 5 cells per well and kept in the appropriate medium during the whole period of observation. Control and KP46 (10 mmol/L)-treated cells were monitored in parallel in a PM S1 incubator (Carl Zeiss MicroImaging) at 37 C and 5% CO 2 using the "mark and find" module of AxioVision (version 4.8.1) image analysis software (19). Recordings started 20 hours after plating and 1 hour after addition of the drug.…”

Phosphorylation of serine 4642 in the COOH-extremity of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

“…3C). Since plectin is dimeric and is able to form oligomers by lateral association (Walko et al, 2011), incompletely phosphorylated plectin oligomers could remain associated with IFs. Moreover, a vimentin-binding site has been identified in the N-terminal ABD of plectin (Sevcik et al, 2004) and plectin interacts with other insoluble proteins than IFs, including microfilaments, microtubules and large membrane protein complexes.…”