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1.
FEBS Lett ; 595(21): 2675-2690, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34626438

RESUMO

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multipolar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.


Assuntos
Proteínas 14-3-3 , Centrossomo , Desmossomos , Mitose , Células HCT116 , Humanos , Fuso Acromático
2.
ESC Heart Fail ; 8(5): 3690-3695, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34432378

RESUMO

Pemphigus is a rare disease characterized by bullous lesions of the skin and mucous membranes. The aetiology is autoimmune and related to the formation of IgG autoantibodies against desmogleins, which are structural proteins of desmosomes that ensure the stability of contacts between cells. Cardiac involvement in patients with pemphigus is poorly documented. We report the data in the literature on this topic and a case of pemphigus-associated autoimmune myocarditis with damage of intercalated disc responding to immunosuppressive therapy. The occurrence of cardiomyopathy with left ventricular dysfunction in patients affected by pemphigus should be appropriately screened with endomyocardial biopsy as it could be the myocardial extension of a potentially reversible autoimmune disorder.


Assuntos
Doenças Autoimunes , Miocardite , Pênfigo , Autoanticorpos , Doenças Autoimunes/complicações , Doenças Autoimunes/diagnóstico , Desmossomos , Humanos , Miocardite/complicações , Miocardite/diagnóstico , Pênfigo/complicações , Pênfigo/diagnóstico
3.
Biomolecules ; 11(6)2021 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-34203070

RESUMO

Desmosomes are intercellular adhesion complexes involved in various aspects of epithelial pathophysiology, including tissue homeostasis, morphogenesis, and disease development. Recent studies have reported that the abnormal expression of various desmosomal components correlates with tumor progression and poor survival. In addition, desmosomes have been shown to act as a signaling platform to regulate the proliferation, invasion, migration, morphogenesis, and apoptosis of cancer cells. The occurrence and progression of head and neck cancer (HNC) is accompanied by abnormal expression of desmosomal components and loss of desmosome structure. However, the role of desmosomal components in the progression of HNC remains controversial. This review aims to provide an overview of recent developments showing the paradoxical roles of desmosomal components in tumor suppression and promotion. It offers valuable insights for HNC diagnosis and therapeutics development.


Assuntos
Desmossomos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Transdução de Sinais , Adesão Celular , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/terapia , Humanos
4.
Sci Transl Med ; 13(603)2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290054

RESUMO

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.


Assuntos
Desmossomos , Células-Tronco Pluripotentes Induzidas , Humanos , Contração Miocárdica , Miócitos Cardíacos , Fenótipo , Engenharia Tecidual
5.
Am J Gastroenterol ; 116(7): 1537-1541, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33955725

RESUMO

INTRODUCTION: We assessed if obesity perturbs the esophageal epithelial barrier function independent of promotion of gastroesophageal reflux (GER). METHODS: Thirty-eight participants were divided into 4 groups: Obesity-/GER-, Obesity+/GER-, Obesity-/GER+, and Obesity+/GER+. Esophageal intercellular space and desmosome density (structural integrity) and fluorescein leak (functional integrity) were measured. RESULTS: The Obesity+/GER- group demonstrated increased intercellular space, reduced desmosome density, and increased fluorescein leak compared with control subjects. These changes were similar but not additive to findings seen in Obesity-/GER + and Obesity+/GER+ patients. DISCUSSION: Central obesity impairs structural and functional integrity of the esophageal barrier independent of GER, likely predisposing to esophageal injury.


Assuntos
Mucosa Esofágica/metabolismo , Espaço Extracelular , Refluxo Gastroesofágico/metabolismo , Obesidade Abdominal/metabolismo , Permeabilidade , Adulto , Idoso , Desmossomos/ultraestrutura , Mucosa Esofágica/patologia , Mucosa Esofágica/ultraestrutura , Feminino , Refluxo Gastroesofágico/complicações , Refluxo Gastroesofágico/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade Abdominal/complicações , Obesidade Abdominal/patologia
6.
J Clin Invest ; 131(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33857019

RESUMO

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Displasia Arritmogênica Ventricular Direita/metabolismo , Complexo do Signalossomo COP9/metabolismo , Desmossomos/metabolismo , Proteólise , Proteoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Displasia Arritmogênica Ventricular Direita/genética , Complexo do Signalossomo COP9/genética , Desmossomos/genética , Desmossomos/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteoma/genética
7.
Dokl Biol Sci ; 496(1): 17-20, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33635484

RESUMO

The excretory system ultrastructure and immunocytochemistry have been investigated in the plerocercoid Pyramicocephalus phocarum. It has been shown that P. phocarum has independent terminal cells, cyrtocytes. The entire canal system is a single undivided syncytium, which includes nephridial funnels of the terminal tubules, and peripheral and central canals. The nephridial funnel and cyrtocyte form a filtration complex of the protonephridial type. In the caudal region, several peripheral canals open into a deep fold of the tegument, the urinary bladder. The excretory pores are separated from the tegument by annular septate desmosomes. There are no cell junctions inside the excretory system. The presence of the F-actin ring and the expression of non-synaptic serotonin in the collar area have been detected in cyrtocytes by immunocytochemistry methods.


Assuntos
Cestoides/ultraestrutura , Desmossomos/genética , Junções Intercelulares/genética , Bexiga Urinária/metabolismo , Actinas/genética , Animais , Cestoides/metabolismo , Cestoides/fisiologia , Regulação da Expressão Gênica/genética , Células Gigantes/metabolismo , Células Gigantes/fisiologia , Junções Intercelulares/metabolismo , Serotonina/genética , Bexiga Urinária/ultraestrutura
9.
Mol Biol Cell ; 32(8): 753-768, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33596089

RESUMO

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal-epithelial-specific Dsc2 knockdown (KD) (Dsc2ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell-cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.


Assuntos
Adesão Celular/fisiologia , Desmocolinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Desmocolinas/genética , Desmocolinas/fisiologia , Desmogleína 2/metabolismo , Caderinas de Desmossomos/metabolismo , Caderinas de Desmossomos/fisiologia , Desmossomos/metabolismo , Humanos , Junções Intercelulares/metabolismo , Mucosa Intestinal , Masculino , Camundongos , Camundongos Knockout
10.
Int J Biol Macromol ; 170: 549-560, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385445

RESUMO

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. However, PKP1 can be also found in the nucleus of several cells. NUPR1 is an intrinsically disordered protein (IDP) that localizes throughout the whole cell, and intervenes in the development and progression of several cancers. In this work, we studied the binding between PKP1 and NUPR1 by using several in vitro biophysical techniques and in cellulo approaches. The interaction occurred with an affinity in the low micromolar range (~10 µM), and involved the participation of at least one of the tryptophan residues of PKP1 (as shown by fluorescence and molecular docking). The binding region of NUPR1, mapped by NMR and molecular modelling, was a polypeptide patch at the 30s region of its sequence. The association between PKP1 and NUPR1 also occurred in cellulo and was localized in the nucleus, as tested by protein ligation assays (PLAs). We hypothesize that NUPR1 plays an active role in carcinogenesis modulating the function of PKP1.


Assuntos
Tatus/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Neoplasias/metabolismo , Placofilinas/metabolismo , Ligação Proteica/fisiologia , Animais , Carcinogênese/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Desmossomos/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Imageamento por Ressonância Magnética/métodos , Masculino , Simulação de Acoplamento Molecular/métodos , Domínios Proteicos/fisiologia , Triptofano/metabolismo
11.
Cancer Res ; 81(6): 1513-1527, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33461973

RESUMO

Ras proteins play a causal role in human cancer by activating multiple pathways that promote cancer growth and invasion. However, little is known about how Ras induces the first diagnostic features of invasion in solid tumors, including loss of epithelial integrity and breaching of the basement membrane (BM). In this study, we found that oncogenic Ras strongly promotes the activation of hepsin, a member of the hepsin/TMPRSS type II transmembrane serine protease family. Mechanistically, the Ras-dependent hepsin activation was mediated via Raf-MEK-ERK signaling, which controlled hepsin protein stability through the heat shock transcription factor-1 stress pathway. In Ras-transformed three-dimensional mammary epithelial culture, ablation of hepsin restored desmosomal cell-cell junctions, hemidesmosomes, and BM integrity and epithelial cohesion. In tumor xenografts harboring mutant KRas, silencing of hepsin increased local invasion concomitantly with accumulation of collagen IV. These findings suggest that hepsin is a critical protease for Ras-dependent tumorigenesis, executing cell-cell and cell-matrix pathologies important for early tumor dissemination. SIGNIFICANCE: These findings identify the cell-surface serine protease hepsin as a potential therapeutic target for its role in oncogenic Ras-mediated deregulation of epithelial cell-cell and cell-matrix interactions and cohesion of epithelial structure.


Assuntos
Neoplasias da Mama/patologia , Células Epiteliais/patologia , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina Endopeptidases/metabolismo , Animais , Membrana Basal/citologia , Membrana Basal/patologia , Mama/patologia , Neoplasias da Mama/genética , Carcinogênese/patologia , Comunicação Celular , Linhagem Celular Tumoral , Colágeno Tipo IV/metabolismo , Desmossomos/patologia , Células Epiteliais/citologia , Feminino , Técnicas de Silenciamento de Genes , Fatores de Transcrição de Choque Térmico/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Mutação , Invasividade Neoplásica/patologia , Cultura Primária de Células , Estabilidade Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Serina Endopeptidases/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Environ Int ; 148: 106382, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472089

RESUMO

Spontaneous preterm birth is a syndrome with clinical and genetic heterogeneity. Few studies have focused on the genetic and epigenetic defects and pathogenic mechanisms associated with premature uterine contraction in spontaneous preterm birth. The objective of this study was to investigate the (epi)genetic variations associated with premature uterine contraction of spontaneous preterm birth. A systems biology approach with an integrated multiomic study was employed. Biobanked pregnancy tissues selected from a pregnancy cohort were subjected to genomic, transcriptomic, methylomic, and proteomic studies, with a focus on genetic loci/genes related to uterine muscle contraction, specifically, genes associated with sarcomeres and desmosomes. Thirteen single nucleotide variations and pathogenic variants were identified in the sarcomere gene, TTN, which encodes the protein Titin, from 146 women with spontaneous preterm labor. Differential expression profiles of five long non-coding RNAs were identified from loci that overlap with four sarcomeric genes. Longitudinally, the long non-coding RNA of gene TPM3 that encodes the protein tropomysin 3 was found to significantly regulate the mRNA of TPM3 in the placenta, compared to maternal blood. The majority of genome methylation profiles related to premature uterine contraction were also identified in the CpG promoters of sarcomeric genes/loci. Differential expression profiles of mRNAs associated with premature uterine contraction showed 22 genes associated with sarcomeres and three with desmosomes. The results demonstrated that premature uterine contraction was associated mainly with pathogenic variants of the TTN gene and with transcriptomic variations of sarcomeric premature uterine contraction genes. This association is likely regulated by epigenetic factors, including methylation and long non-coding RNAs.


Assuntos
Trabalho de Parto Prematuro , Nascimento Prematuro , Desmossomos , Feminino , Humanos , Recém-Nascido , Trabalho de Parto Prematuro/genética , Gravidez , Proteômica , Sarcômeros/genética
13.
J Invest Dermatol ; 141(5): 1219-1229.e11, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33098828

RESUMO

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules.


Assuntos
Proteínas de Ligação a Calmodulina/fisiologia , Desmossomos/fisiologia , Proteínas dos Microfilamentos/fisiologia , Animais , Caderinas/química , Cálcio/metabolismo , Adesão Celular , Plasticidade Celular , Células Cultivadas , Desmogleína 3/metabolismo , Desmossomos/química , Humanos , Camundongos
14.
Trends Cardiovasc Med ; 31(7): 395-402, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-32738304

RESUMO

Arrhythmogenic cardiomyopathy (ACM) is a familial disease, with approximately 60% of patients displaying a pathogenic variant. The majority of genes linked to ACM code for components of the desmosome: plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2), plakoglobin (JUP) and desmoplakin (DSP). Genetic variants involving the desmosomes are known to cause dysfunction of cell-to-cell adhesions and intercellular gap junctions. In turn, this may result in failure to mechanically hold together the cardiomyocytes, fibrofatty myocardial replacement, cardiac conduction delay and ventricular arrhythmias. It is becoming clearer that pathogenic variants in desmosomal genes such as PKP2 are not only responsible for a mechanical dysfunction of the intercalated disc (ID), but are also the cause of various pro-arrhythmic mechanisms. In this review, we discuss in detail the different molecular interactions associated with desmosomal pathogenic variants, and their contribution to various ACM phenotypes.


Assuntos
Displasia Arritmogênica Ventricular Direita , Desmossomos , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/genética , Displasia Arritmogênica Ventricular Direita/diagnóstico , Displasia Arritmogênica Ventricular Direita/genética , Desmossomos/genética , Humanos , Miocárdio , Placofilinas/genética
15.
Acta Physiol (Oxf) ; 231(4): e13609, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33354837

RESUMO

AIM: Desmoplakin (Dp) is a crucial component of the desmosome, a supramolecular cell junction complex anchoring intermediate filaments. The mechanisms how Dp modulates cell-cell adhesion are only partially understood. Here, we studied the impact of Dp on the function of desmosomal adhesion molecules, desmosome turnover and intercellular adhesion. METHODS: CRISPR/Cas9 was used for gene editing of human keratinocytes which were characterized by Western blot and immunostaining. Desmosomal ultrastructure and function were assessed by electron microscopy and cell adhesion assays. Single molecule binding properties and localization of desmosomal cadherins were studied by atomic force microscopy and super-resolution imaging. RESULTS: Knockout (ko) of Dp impaired cell cohesion to drastically higher extents as ko of another desmosomal protein, plakoglobin (Pg). In contrast to Pg ko, desmosomes were completely absent in Dp ko. Binding properties of the desmosomal adhesion molecules desmocollin (Dsc) 3 and desmoglein (Dsg) 3 remained unaltered under loss of Dp. Dp was required for assembling desmosomal cadherins into large clusters, as Dsg2 and Dsc3, adhesion molecules primarily localized within desmosomes, were redistributed into small puncta in the cell membrane of Dp ko cells. Additional silencing of desmosomal cadherins in Dp ko did not further increase loss of intercellular adhesion. CONCLUSION: Our data demonstrate that Dp is essential for desmosome formation but does not influence intercellular adhesion on the level of individual cadherin binding properties. Rather, macro-clustering of desmosomal adhesion molecules through Dp is crucial. These results may help to better understand severe diseases which are caused by Dp dysfunction.


Assuntos
Caderinas , Desmossomos , Adesão Celular , Análise por Conglomerados , Desmogleínas , Desmoplaquinas , Humanos
16.
Br J Dermatol ; 184(4): 596-605, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32593191

RESUMO

The desmosome is a type of intercellular junction found in epithelial cells, cardiomyocytes and other specialized cell types. Composed of a network of transmembranous cadherins and intracellular armadillo, plakin and other proteins, desmosomes contribute to cell-cell adhesion, signalling, development and differentiation. Mutations in genes encoding desmosomal proteins result in a spectrum of erosive skin and mucosal phenotypes that also may affect hair or heart. This review summarizes the molecular pathology and phenotypes associated with desmosomal dysfunction with a focus on inherited disorders that involve the skin/hair, as well as associated extracutaneous pathologies. We reviewed the relevant literature to collate studies of pathogenic human mutations in desmosomes that have been reported over the last 25 years. Mutations in 12 different desmosome genes have been documented, with mutations in nine genes affecting the skin/mucous membranes (DSG1, DSG3, DSC2, DSC3, JUP, PKP1, DSP, CDSN, PERP) and eight resulting in hair abnormalities (DSG4, DSC2, DSC3, JUP, PKP1, DSP, CDSN, PERP). Mutations in three genes can result in cardiocutaneous syndromes (DSC2, JUP, DSP), although mutations have been described in five genes in inherited heart disorders that may lack any dermatological manifestations (DSG2, DSC2, JUP, PKP2, DSP). Understanding the diverse nature of these clinical phenotypes, as well as the desmosome gene mutation(s), has clinical value in managing and counselling patients, as well as demonstrating the biological role and activity of specific components of desmosomes in skin and other tissues.


Assuntos
Desmossomos , Pele/patologia , Caderinas , Desmogleínas/genética , Desmossomos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Mutação , Fenótipo
17.
Proc Natl Acad Sci U S A ; 117(49): 31157-31165, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229577

RESUMO

We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a "bait" protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2ß1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.


Assuntos
Caderinas/genética , Efrina-B1/genética , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Caderinas/ultraestrutura , Adesão Celular/genética , Citoplasma/genética , Citoplasma/ultraestrutura , Desmocolinas , Desmogleína 2/genética , Desmogleína 2/ultraestrutura , Desmoplaquinas/genética , Desmoplaquinas/ultraestrutura , Desmossomos/genética , Desmossomos/ultraestrutura , Efrina-B1/ultraestrutura , Humanos , Integrinas/genética , Integrinas/ultraestrutura , Microscopia de Força Atômica , Domínios Proteicos/genética , Imagem Individual de Molécula
19.
Cell Adh Migr ; 14(1): 195-203, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33016205

RESUMO

To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.


Assuntos
Movimento Celular , Desmossomos/metabolismo , Galectina 4/metabolismo , Pseudópodes/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Desmossomos/ultraestrutura , Galectina 4/genética , Humanos , Pseudópodes/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(44): 27132-27140, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33067392

RESUMO

Desmosomes are cell-cell junctions that link tissue cells experiencing intense mechanical stress. Although the structure of the desmosomal cadherins is known, the desmosome architecture-which is essential for mediating numerous functions-remains elusive. Here, we recorded cryo-electron tomograms (cryo-ET) in which individual cadherins can be discerned; they appear variable in shape, spacing, and tilt with respect to the membrane. The resulting sub-tomogram average reaches a resolution of ∼26 Å, limited by the inherent flexibility of desmosomes. To address this challenge typical of dynamic biological assemblies, we combine sub-tomogram averaging with atomistic molecular dynamics (MD) simulations. We generate models of possible cadherin arrangements and perform an in silico screening according to biophysical and structural properties extracted from MD simulation trajectories. We find a truss-like arrangement of cadherins that resembles the characteristic footprint seen in the electron micrograph. The resulting model of the desmosomal architecture explains their unique biophysical properties and strength.


Assuntos
Desmossomos/química , Tomografia com Microscopia Eletrônica/métodos , Caderinas/química , Caderinas/metabolismo , Desmossomos/metabolismo , Desmossomos/fisiologia , Humanos , Junções Intercelulares , Simulação de Dinâmica Molecular
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