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Desmosomes mediate cell-cell adhesion and are prevalent in tissues under mechanical stress. However, their detailed structural characterization is not available. Here, we characterized the molecular architecture of the desmosomal outer dense plaque (ODP) using Bayesian integrative structural modeling via the Integrative Modeling Platform. Starting principally from the structural interpretation of a cryo-electron tomography (cryo-ET) map of the ODP, we integrated information from x-ray crystallography, an immuno-electron microscopy study, biochemical assays, in silico predictions of transmembrane and disordered regions, homology modeling, and stereochemistry information. The integrative structure was validated by information from imaging, tomography, and biochemical studies that were not used in modeling. The ODP resembles a densely packed cylinder with a plakophilin (PKP) layer and a plakoglobin (PG) layer; the desmosomal cadherins and PKP span these two layers. Our integrative approach allowed us to localize disordered regions, such as the N-terminus of PKP and the C-terminus of PG. We refined previous protein-protein interactions between desmosomal proteins and provided possible structural hypotheses for defective cell-cell adhesion in several diseases by mapping disease-related mutations on the structure. Finally, we point to features of the structure that could confer resilience to mechanical stress. Our model provides a basis for generating experimentally verifiable hypotheses on the structure and function of desmosomal proteins in normal and disease states., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare and potentially life-threatening disorder of the heart. The clinical spectrum of ARVC includes myocyte loss and fibro-fatty tissue replacement. With the progress of ARVC, the patient can present serious ventricular arrhythmias, heart failure, and even sudden cardiac death. Previous studies have demonstrated that desmosomes and intermediate junctions play a crucial role in the generation and development of ARVC. In this study, we enrolled a Chinese patient with suspicious ARVC. The patient suffered from right ventricular enlargement and less thickening of right ventricular wall. ECG record showed an epsilon wave. However, there was no obvious symptom in his parents. After whole-exome sequencing and data filtering, we identified a de novo mutation (c.1729C>T/p.R577C) of junction plakoglobin ( JUP ) in this patient. Bioinformatics programs predicted that this mutation was deleterious. Western blot revealed that, compared to cells transfected with WT plasmids, the expressions of desmoglein 2 ( DSG2 ) and Connexin 43 were decreased overtly in cells transfected with the mutant plasmid. Previous studies have proven that the reduction of DSG2 and Connexin 43 may disturb the stability of desmosomes. In this research, we reported a novel de novo mutation (c.1729C>T/p.R577C) of JUP in a Chinese patient with suspicious ARVC. Functional research further confirmed the pathogenicity of this novel mutation. Our study expanded the spectrum of JUP mutations and may contribute to the genetic diagnosis and counseling of patients with ARVC.

Arrhythmogenic cardiomyopathy (AC) is associated with mutations in genes encoding intercalated disc proteins and ultimately results in sudden cardiac death. A subset of patients with AC have the autosomal recessive cardiocutaneous disorder Naxos disease, which is caused by a 2-base pair deletion in the plakoglobin-encoding gene JUP that results in a truncated protein with reduced expression. In mice, cardiomyocyte-specific plakoglobin deficiency recapitulates many aspects of human AC, and overexpression of the truncated Naxos-associated plakoglobin also results in an AC-like phenotype; therefore, it is unclear whether Naxos disease results from loss or gain of function consequent to the plakoglobin mutation. Here, we generated 2 knockin mouse models in which endogenous Jup was engineered to express the Naxos-associated form of plakoglobin. In one model, Naxos plakoglobin bypassed the nonsense-mediated mRNA decay pathway, resulting in normal levels of the truncated plakoglobin. Moreover, restoration of Naxos plakoglobin to WT levels resulted in normal heart function. Together, these data indicate that a gain of function in the truncated form of the protein does not underlie the clinical phenotype of patients with Naxos disease and instead suggest that insufficiency of the truncated Naxos plakoglobin accounts for disease manifestation. Moreover, these results suggest that increasing levels of truncated or WT plakoglobin has potential as a therapeutic approach to Naxos disease.

Plakoglobin (PG, γ-Catenin, JUP), a member of the armadillo protein family and close homolog of β-catenin, functions to link cell surface cadherin molecules with the cytoskeleton. PG is the only junctional component found in both desmosomes and adherens junctions and thus plays a critical role in the regulation of cell-cell adhesion. Similar to β-catenin, PG is able to interact with components of the Wnt signaling pathway and directly affect gene expression by binding with LEF/TCF transcription factors. In addition, it has been proposed that PG functions primarily as a competitive inhibitor of β-catenin transcriptional activity by sequestering LEF/TCF. Compared to β-catenin, the contribution of PG as a transcriptional regulator in either physiological or pathological conditions is poorly understood. There is increasing clinical interest in PG as both a structural protein as well as a signaling molecule as mutations have been identified in the human PG gene that cause Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) and cutaneous syndromes. This review will discuss the connection between altered cell adhesion and gene expression and its contribution to disease pathogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Plakoglobin (gamma-catenin) is a homolog of beta-catenin with similar dual adhesive and signaling functions. The adhesive function of these proteins is mediated by their interactions with cadherins, whereas their signaling activity is regulated by association with various intracellular partners. In this respect, beta-catenin has a well-defined oncogenic activity through its role in the Wnt signaling pathway, whereas plakoglobin acts as a tumor/metastasis suppressor through mechanisms that remain unclear. We previously expressed plakoglobin in SCC9 squamous carcinoma cells (SCC9-P) and observed a mesenchymal-to-epidermoid transition. Comparison of the protein and RNA profiles of parental SCC9 cells and SCC9-P transfectants identified various differentially expressed proteins and transcripts, including the nonmetastatic protein 23 (Nm23). In this study, we show that Nm23-H1 mRNA and Nm23-H2 protein are increased after plakoglobin expression. Coimmunoprecipitation and confocal microscopy studies using SCC9-P and various epithelial cell lines with endogenous plakoglobin expression revealed that Nm23 interacts with plakoglobin, cadherins and alpha-catenin. Furthermore, Nm23-H2 is the primary isoform involved in these interactions, which occur prominently in the cytoskeleton-associated pool of cellular proteins. In addition, we show that plakoglobin-Nm23 interaction requires the N-terminal (alpha-catenin interacting) domain of plakoglobin. Our data suggest that by increasing the expression and stability of Nm23, plakoglobin has a role in regulating the metastasis suppressor activity of Nm23, which may further provide a potential mechanism for the tumor/metastasis suppressor function of plakoglobin itself.

O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion.

Desmosomes are adhesive intercellular junctions prominent in the skin and heart. Loss of desmosome function is associated with severe congenital and acquired disorders characterized by tissue fragility. Pemphigus vulgaris (PV) is an autoimmune disorder in which antibodies are directed against the desmosomal adhesion molecule Dsg3, resulting in severe mucosal erosions and epidermal blistering. To define the mechanisms by which Dsg3 autoantibodies disrupt keratinocyte adhesion, the fate of PV IgG and various desmosomal components was monitored in primary human keratinocytes exposed to PV patient IgG. PV IgG initially bound to keratinocyte cell surfaces and colocalized with desmosomal markers. Within 6 h after PV IgG binding to Dsg3, electron microscopy revealed that desmosomes were dramatically disrupted and keratinocyte adhesion was severely compromised. Immunofluorescence analysis indicated that PV IgG and Dsg3 were rapidly internalized from the cell surface in a complex with plakoglobin but not desmoplakin. Dsg3 internalization was associated with retraction of keratin filaments from cell-cell borders. Furthermore, the internalized PV IgG-Dsg3 complex colocalized with markers for both endosomes and lysosomes, suggesting that Dsg3 was targeted for degradation. Consistent with this possibility, biotinylation experiments demonstrated that soluble Dsg3 cell surface pools were rapidly depleted followed by loss of detergent-insoluble Dsg3. These findings demonstrate that Dsg3 endocytosis, keratin filament retraction, and the loss of keratinocyte cell-cell adhesion are coordinated responses to PV IgG.