Missense Mutations in Desmoplakin Plakin Repeat Domains Have Dramatic Effects on Domain Structure and Function.

Plakin repeat domains (PRDs) are globular modules that mediate the interaction of plakin proteins with the intermediate filament (IF) cytoskeleton. These associations are vital for maintaining tissue integrity in cardiac muscle and epithelial tissues. PRDs are subject to mutations that give rise to cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy, characterised by ventricular arrhythmias and associated with an increased risk of sudden heart failure, and skin blistering diseases. Herein, we have examined the functional and structural effects of 12 disease-linked missense mutations, identified from the human gene mutation database, on the PRDs of the desmosomal protein desmoplakin. Five mutations (G2056R and E2193K in PRD-A, G2338R and G2375R in PRD-B and G2647D in PRD-C) rendered their respective PRD proteins either fully or partially insoluble following expression in bacterial cells. Each of the residues affected are conserved across plakin family members, inferring a crucial role in maintaining the structural integrity of the PRD. In transfected HeLa cells, the mutation G2375R adversely affected the targeting of a desmoplakin C-terminal construct containing all three PRDs to vimentin IFs. The deletion of PRD-B and PRD-C from the construct compromised its targeting to vimentin. Bioinformatic and structural modelling approaches provided multiple mechanisms by which the disease-causing mutations could potentially destabilise PRD structure and compromise cytoskeletal linkages. Overall, our data highlight potential molecular mechanisms underlying pathogenic missense mutations and could pave the way for informing novel curative interventions targeting cardiomyopathies and skin blistering disorders.

Desmosomal protein structure and function and the impact of disease-causing mutations.

In this graphical review we focus on the structural characteristics of desmosomal proteins, their interactions with each other and with the intermediate filament cytoskeleton. The wealth of structural information that is now available allows predictions to be made about the pathogenic effect of disease-causing mutations. We have selected representative examples of missense mutations that are buried, semi-buried or surface exposed, and demonstrate how such variants could affect the structural fold of desmosomal proteins that are expressed in the heart. We explain how such alterations could compromise desmosomal adhesion, resulting in life threatening diseases including arrhythmogenic right ventricular cardiomyopathy.

Molecular mechanism of intermediate filament recognition by plakin proteins.

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.

Binding of the periplakin linker requires vimentin acidic residues D176 and E187.

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.

Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon.

Krüppel-like factor 5 (KLF5) is a member of the zinc finger family of transcription factors that regulates homeostasis of the intestinal epithelium. Previous studies suggested an indispensable role of KLF5 in maintaining intestinal barrier function. In the current study, we investigated the mechanisms by which KLF5 regulates colonic barrier function in vivo and in vitro. We used an inducible and a constitutive intestine-specific Klf5 knockout mouse models (Villin-CreERT2;Klf5fl/fl designated as Klf5ΔIND and Villin-Cre;Klf5fl/fl as Klf5ΔIS ) and studied an inducible KLF5 knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe KLF5ΔIND). Specific knockout of Klf5 in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in Dsg2, which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy showed alterations of desmosomal morphology in both KLF5 knockdown Caco-2 BBe cells and Klf5 knockout mouse colonic tissues. Inducible knockdown of KLF5 in Caco-2BBe cells grown on Transwell plates led to impaired barrier function as evidenced by decreased transepithelial electrical resistance and increased paracellular permeability to fluorescein isothiocyanate-4 kDa dextran. Furthermore, DSG2 was significantly decreased in KLF5 knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by KLF5 knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after KLF5 knockdown. In combination with chromatin immunoprecipitation analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of DSG2, a gene encoding a major component of desmosome structures.NEW & NOTEWORTHY The study is original research on the direct function of a Krüppel-like factor on intestinal barrier function, which is commonly exerted by cell junctions, including tight junctions, adherens junctions, and desmosomes. Numerous previous studies were focused on tight junctions and adherens junctions. However, this study provided a new perspective on how the intestinal barrier function is regulated by KLF5 through DSG2, a component of desmosome complexes.

Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin.

Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

Sequence-specific 1H, 13C and 15N backbone resonance assignments of the plakin repeat domain of human envoplakin.

The plakin repeat domain is a distinctive hallmark of the plakin superfamily of proteins, which are found within all epithelial tissues. Plakin repeat domains mediate the interactions of these proteins with the cell cytoskeleton and are critical for the maintenance of tissue integrity. Despite their biological importance, no solution state resonance assignments are available for any homologue. Here we report the essentially complete (1)H, (13)C and (15)N backbone chemical shift assignments of the singular 22 kDa plakin repeat domain of human envoplakin, providing the means to investigate its interactions with ligands including intermediate filaments.

Hinged plakin domains provide specialized degrees of articulation in envoplakin, periplakin and desmoplakin.

Envoplakin, periplakin and desmoplakin are cytoskeletal proteins that provide structural integrity within the skin and heart by resisting shear forces. Here we reveal the nature of unique hinges within their plakin domains that provides divergent degrees of flexibility between rigid long and short arms composed of spectrin repeats. The range of mobility of the two arms about the hinge is revealed by applying the ensemble optimization method to small-angle X-ray scattering data. Envoplakin and periplakin adopt 'L' shaped conformations exhibiting a 'helicopter propeller'-like mobility about the hinge. By contrast desmoplakin exhibits essentially unrestricted mobility by 'jack-knifing' about the hinge. Thus the diversity of molecular jointing that can occur about plakin hinges includes 'L' shaped bends, 'U' turns and fully extended 'I' orientations between rigid blocks of spectrin repeats. This establishes specialised hinges in plakin domains as a key source of flexibility that may allow sweeping of cellular spaces during assembly of cellular structures and could impart adaptability, so preventing irreversible damage to desmosomes and the cell cytoskeleton upon exposure to mechanical stress.

Mechanistic basis of desmosome-targeted diseases.

Desmosomes are dynamic junctions between cells that maintain the structural integrity of skin and heart tissues by withstanding shear forces. Mutations in component genes cause life-threatening conditions including arrhythmogenic right ventricular cardiomyopathy, and desmosomal proteins are targeted by pathogenic autoantibodies in skin blistering diseases such as pemphigus. Here, we review a set of newly discovered pathogenic alterations and discuss the structural repercussions of debilitating mutations on desmosomal proteins. The architectures of native desmosomal assemblies have been visualized by cryo-electron microscopy and cryo-electron tomography, and the network of protein domain interactions is becoming apparent. Plakophilin and desmoplakin mutations have been discovered to alter binding interfaces, structures, and stabilities of folded domains that have been resolved by X-ray crystallography and NMR spectroscopy. The flexibility within desmoplakin has been revealed by small-angle X-ray scattering and fluorescence assays, explaining how mechanical stresses are accommodated. These studies have shown that the structural and functional consequences of desmosomal mutations can now begin to be understood at multiple levels of spatial and temporal resolution. This review discusses the recent structural insights and raises the possibility of using modeling for mechanism-based diagnosis of how deleterious mutations alter the integrity of solid tissues.

The nonlinear structure of the desmoplakin plakin domain and the effects of cardiomyopathy-linked mutations.

Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. The desmoplakin N-terminal region is a 1056-amino-acid sequence of unknown structure. It mediates interactions with other desmosomal proteins, is found in a variety of plakin proteins, and spans what has been termed the "plakin domain," which includes residues 180-1022 and consists of six spectrin repeats (SRs) and an Src homology 3 domain. Herein we elucidate the architecture of desmoplakin's plakin domain, as well as its constituent tandem SRs. Small-angle X-ray scattering analysis shows that the entire plakin domain has an "L" shape, with a long arm and a short arm held at a perpendicular angle. The long arm is 24.0 nm long and accommodates four stably folded SRs arranged in tandem. In contrast, the short arm is 17.9 nm in length and accommodates two independently folded repeats and an extended C-terminus. We show that mutations linked to arrhythmogenic right ventricular cardiomyopathy (K470E and R808C) cause local conformational alterations, while the overall folded structure is maintained. This provides the first structural and mechanistic insights into an entire plakin domain and provides a basis for understanding the critical role of desmoplakin in desmosome function.

The desmoglein-specific cytoplasmic region is intrinsically disordered in solution and interacts with multiple desmosomal protein partners.

The desmoglein-specific cytoplasmic region (DSCR) is a conserved region of unknown structure and function that uniquely defines the desmoglein family of cell adhesion molecules. It is the site of caspase cleavage during apoptosis, and its mutation is linked to cardiomyopathy. Here, we reveal that a 276-residue DSCR construct of human desmoglein 1 is intrinsically disordered and forms an interaction hub for desmosomal proteins. In solution, it contains 6.5% helical and 10.3% beta-strand structure based on circular dichroism spectroscopy. A single monomeric state with a predominantly unfolded structure is found by size-exclusion chromatography and analytical ultracentrifugation. Thermal stability assays and nuclear magnetic resonance spectroscopy reveal a nonglobular structure under a range of solution conditions. However, the introduction of detergent micelles increases structure to 18% helical and 16% beta-strand character, suggesting an inducible structure. The DSCR exhibits weak but specific interactions with plakoglobin, the plakin domain of desmoplakin, plakophilin 1, and the cytoplasmic domain of desmocollin 1. The desmoglein 1 membrane proximal region also interacts with all four DSCR ligands, strongly with plakoglobin and plakophilin and more weakly with desmoplakin and desmocollin 1. Thus, the DSCR is an intrinsically disordered functional domain with an inducible structure that, along with the membrane proximal region, forms a flexible scaffold for cytoplasmic assembly at the desmosome.

Desmosome structure, composition and function.

Desmosomes are intercellular junctions of epithelia and cardiac muscle. They resist mechanical stress because they adopt a strongly adhesive state in which they are said to be hyper-adhesive and which distinguishes them from other intercellular junctions; desmosomes are specialised for strong adhesion and their failure can result in diseases of the skin and heart. They are also dynamic structures whose adhesiveness can switch between high and low affinity adhesive states during processes such as embryonic development and wound healing, the switching being signalled by protein kinase C. Desmosomes may also act as signalling centres, regulating the availability of signalling molecules and thereby participating in fundamental processes such as cell proliferation, differentiation and morphogenesis. Here we consider the structure, composition and function of desmosomes, and their role in embryonic development and disease.

Regulation of desmocollin gene expression in the epidermis: CCAAT/enhancer-binding proteins modulate early and late events in keratinocyte differentiation.

Desmocollins (Dscs) are desmosomal cadherins that exhibit differentiation-specific patterns of expression in the epidermis. Dsc3 expression is strongest in basal cell layers, whereas Dsc1 is largely confined to upper, terminally differentiating strata. To understand better the processes by which Dsc expression is regulated in the epidermis, we have isolated Dsc3 and Dsc1 5'-flanking DNAs and analysed their activity in primary keratinocytes. In the present study, we found that transcription factors of the CCAAT/enhancer-binding protein family play a role in the regulation of expression of both Dscs and, in so doing, implicate this class of transcription factors in both early and late events in keratinocyte differentiation. We show that Dscs are differentially regulated by C/EBP (CCAAT/enhancer-binding protein) family members, with Dsc3 expression being activated by C/EBPbeta but not C/EBPalpha, and the reverse being the case for Dsc1. Expression of both Dscs is activated by another family member, C/EBPdelta. These results show for the first time how desmosomal cadherin gene expression is regulated and provide a mechanism for the control of other differentiation-specific genes in the epidermis.