A Nuclear Function for Plakophilin-1 in the DNA Damage Response?

Plakophilins are proteins of the desmosomal plaque. Based on the observation that plakophilins localize not only to desmosomes but also to the cytoplasm and nucleus, additional functions in cell signaling have been proposed. In this issue, Sobolik-Delmaire et al. address the nuclear function of Plakophilin-1. The authors show that Plakophilin-1 interacts with ssDNA in vitro and may have a function in protecting cells from DNA damage

Defining desmosomal plakophilin-3 interactions

Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP–PKP3 interaction sites. This finding might explain how lateral DP–PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes

Plakophilin 1 stimulates translation by promoting eIF4A1 activity

p120 armadillo protein plakophilin 1 binds to eukaryotic translation factor eIF4A1, recruiting it into cap-binding complexes and stimulating translation

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered rhoa signalling, epithelial polarization and cytokinesis

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling

Tutorial introdutório do Port4NooJ

Background: Plakophilin2 (PKP2) is a desmosome-related protein with numerous armadillo repeats and has been linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). Fatal arrhythmias resulting in sudden death also occur in the absence of morphologic cardiac abnormalities at autopsy, and have been linked to ion channel mutations in a subset of cases, but so far not to PKP2.Methods and Results: We sequenced all 14 exons of PKP2 in DNA extracted from postmortem heart tissues of 25 patients dying from ARVC and 25 from sudden unexpected death with negative autopsy (SUDNA). the primers were designed using the Primer Express 3.0 software. Direct sequencing for both sense and antisense strands was performed with a BigDye Terminator DNA sequencing kit on a 3130XL Genetic Analyzer. Mutation damage prediction was made using Mutation Taster, Polyphen and SIFT software. in 6 of the 25 ARVC samples, 6 PKP2 mutations were identified, 4 of which were likely significant, and 3 of which were novel (p.N641del, p.L64PfsX22, p.G269R). in 6 of the 25 cases of SUDNA samples, 6 PKP2 mutations were identified, 3 of which were likely significant, and 4 of which were not previously described (p.P665S, p.Y217TfsX45, p.E540, p.S615T).Conclusions: PKP2 mutations are not specific for ARVC and may result in SUDNA. the link between ARVC and desmosomal mutations may not be causal but related to an association between defective desmosomal proteins and arrhythmias. (Circ J 2012; 76: 189-194

The coiled coil of in vitro assembled keratin filaments is a beterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression

Abstract. Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil. Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the a-helical rod domain of both keratins to a cysteine. The mutations lie at position-36 of the rod in a "d " position of the heptad repeat pattern, and thus air oxidation can introduce a zerolength cystine cross-link. Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration. Heterodimers with a cystine cross-link wer

A synthetic peptide representing the consensus sequence motif at the carboxy-terminal end of the rod domain inhibits intermediate filament assembly and disassembles preformed filaments

sequent IF assembly. Filaments displayed a rough surface and variable diameters with a substantial amount present in unravelled form. At higher peptide concentration (50-100-fold molar excess), IF formation was completely inhibited and instead only loose aggregates of "globular " particles were formed. The peptide also influenced preformed keratin IF in a dose-dependent manner. While a three-fold molar excess was sufficient to cause partial fragmentation of IF, a 50-fold molar excess caused complete disassembly within 5 min. Loosely associated protofibrils, short needlelike IF fragments, and aggregates of globular particles were detected. The motif peptide also caused the disassem-Abstract. All intermediate filament (IF) proteins share bly of filaments formed by desmin, a type III IF proa highly conserved sequence motif at the COOH-ter- tein. Peptide concentrations and incubation times remina

Desmosomes as signaling hubs in the regulation of cell behavior

Desmosomes are intercellular junctions, which preserve tissue integrity during homeostatic and stress conditions. These functions rely on their unique structural properties, which enable them to respond to context-dependent signals and transmit them to change cell behavior. Desmosome composition and size vary depending on tissue specific expression and differentiation state. Their constituent proteins are highly regulated by posttranslational modifications that control their function in the desmosome itself and in addition regulate a multitude of desmosome-independent functions. This review will summarize our current knowledge how signaling pathways that control epithelial shape, polarity and function regulate desmosomes and how desmosomal proteins transduce these signals to modulate cell behavior.Publikationsfonds ML