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Localization of occludin peptides. Divalent cation depletion was used to dissemble tight junction (TJ) structures of polarized, confluent T84 cell monolayers. Bait peptide (200 M O-A:101-121* or O-B: 210-228*) or media used for peptide addition (CTRL) was added at the time of Ca 2 repletion. After a 6-or 24-h incubation, monolayers were washed free of unbound peptide, fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, and prepared for fluorescence microscopy. Distribution of bound bait peptides was determined by staining with FITC-conjugated streptavidin (green), whereas Alexa 568phalloidin was used to highlight the F-actin architecture (red). Scale bar, 10 m.
Source publication
Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101-121 and O-B:210-228, respectively, to examine potential molecular interactions be...
Contexts in source publication
Context 1
... of O-A:101-121* to T84 monolayers at the time of Ca 2 repletion did not affect the recovery of F-actin organi- zation (demonstrated by Alexa568-phalloidin staining) at either 6 or 24 h compared with cells exposed to control media ( Figure 3). Application of O-B:210 -228*, however, showed that this peptide interacted strongly with T84 cells, forming aggregates of biotin label that increased over time and localized to sites where F-actin reorganization was re- tarded ( Figure 3). ...
Context 2
... of O-A:101-121* to T84 monolayers at the time of Ca 2 repletion did not affect the recovery of F-actin organi- zation (demonstrated by Alexa568-phalloidin staining) at either 6 or 24 h compared with cells exposed to control media ( Figure 3). Application of O-B:210 -228*, however, showed that this peptide interacted strongly with T84 cells, forming aggregates of biotin label that increased over time and localized to sites where F-actin reorganization was re- tarded ( Figure 3). The lack of O-A:101-121* association with T84 monolayers but the strong association of O-B:210 -228* with these cells was consistent with results obtained in protein labeling studies (Figure 2). ...
Context 3
... of O-B:210 -228* at the time of Ca 2 repletion did not dramatically influence the recovery of polarized F-actin organization at 6 or 24 h ( Figure 4); however, minor differences in actin organization were observed, in agree- ment of previous studies (Figure 3 (Table 1), failed to affect the reorganization of TJ structures when added at the time of Ca 2 repletion (unpublished data). Structural prediction (http://www.cmpharm.ucsf.edu/nomi/nnpredict.html) of O-B:210 -228 suggested no predictable structure outside of an extended conformation for residues YAL near the N-terminus of the SQIYALCNQFYTPAATGLYVD peptide. ...
Context 4
... O-B:210 -228* strongly associated with T84 cells and was observed to form complexes at the surface of T84 cells that increased in size over time (comparing the 0-, 6-, and 24-h time points). Micrographs (Figures 3 and 4) ob- tained at focal planes of the apical neck region of T84 cell monolayers showed that TJ proteins claudin-1, occludin, ...
Citations
... The independent prediction of small solute transport by a single claudin, www.nature.com/scientificreports/ claudin-2, is especially noteworthy in view of the complexity of the TJ system and the heterotypic interactions between different TJ proteins 46 . The significance of molecular determinants of peritoneal permeability has recently been demonstrated for the peritoneal water selective AQP1 channel, which in mice exerts about 50% of water transfer 47 . ...
Next to the skin, the peritoneum is the largest human organ, essentially involved in abdominal health and disease states, but information on peritoneal paracellular tight junctions and transcellular channels and transporters relative to peritoneal transmembrane transport is scant. We studied their peritoneal localization and quantity by immunohistochemistry and confocal microscopy in health, in chronic kidney disease (CKD) and on peritoneal dialysis (PD), with the latter allowing for functional characterizations, in a total of 93 individuals (0–75 years). Claudin-1 to -5, and -15, zonula occludens-1, occludin and tricellulin, SGLT1, PiT1/SLC20A1 and ENaC were consistently detected in mesothelial and arteriolar endothelial cells, with age dependent differences for mesothelial claudin-1 and arteriolar claudin-2/3. In CKD mesothelial claudin-1 and arteriolar claudin-2 and -3 were more abundant. Peritonea from PD patients exhibited increased mesothelial and arteriolar claudin-1 and mesothelial claudin-2 abundance and reduced mesothelial and arteriolar claudin-3 and arteriolar ENaC. Transperitoneal creatinine and glucose transport correlated with pore forming arteriolar claudin-2 and mesothelial claudin-4/-15, and creatinine transport with mesothelial sodium/phosphate cotransporter PiT1/SLC20A1. In multivariable analysis, claudin-2 independently predicted the peritoneal transport rates. In conclusion, tight junction, transcellular transporter and channel proteins are consistently expressed in peritoneal mesothelial and endothelial cells with minor variations across age groups, specific modifications by CKD and PD and distinct associations with transperitoneal creatinine and glucose transport rates. The latter deserve experimental studies to demonstrate mechanistic links.
Clinical Trial registration: The study was performed according to the Declaration of Helsinki and is registered at www.clinicaltrials.gov (NCT01893710).
... Peptides derived from the extracellular loops (EL) of OCLN were shown to bind to OCLN, indicating that transcellular homotypic OCLN interactions between cells occur (19)(20)(21)(22)(23)(24). In order to prevent the 175 potential interactions of mOCLN with endothelial OCLN, we designed peptides mimicking the extracellular loop 1 (EL1) or 2 (EL2) of the human OCLN and associated scramble controls (scrEL1 and scrEL2 respectively; Figure 3A) based on previous literature (( [19][20][21][22][23][24]; see Material & Methods for details). ...
... Peptides derived from the extracellular loops (EL) of OCLN were shown to bind to OCLN, indicating that transcellular homotypic OCLN interactions between cells occur (19)(20)(21)(22)(23)(24). In order to prevent the 175 potential interactions of mOCLN with endothelial OCLN, we designed peptides mimicking the extracellular loop 1 (EL1) or 2 (EL2) of the human OCLN and associated scramble controls (scrEL1 and scrEL2 respectively; Figure 3A) based on previous literature (( [19][20][21][22][23][24]; see Material & Methods for details). None of our peptides significantly changed hCMEC/D3 permeability in vitro ( Figure 3B). ...
Transmigration of circulating monocytes from the bloodstream toward the central nervous system (CNS) represents a hallmark of neuroinflammation and plays an important role during viral encephalitis and HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in monocyte transmigration through endothelia has been extensively studied, but how monocytes locally unzip tight junction-associated proteins (TJAPs) of the endothelium composing the neurovascular unit (NVU) to reach the CNS remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through cerebral microvessel endothelial cells. Silencing monocytic OCLN (mOCLN) impairs monocyte transmigration, while mOCLN overexpression increases transmigration. Using high-resolution live cell imaging, we observed that mOCLN clusters at the monocyte-endothelium interface during the transmigration process, forming a transient ring of mOCLN at the site of diapedesis. Furthermore, we designed OCLN-derived peptides targeting its extracellular loop (EL) 1 or 2 to prevent potential trans-homotypic interactions of mOCLN with endothelial OCLN. We found that transmigration of human monocytes was significantly inhibited upon treatment with the EL2 peptide in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and here, we showed that the treatment of transmigrating monocytes with the EL2 peptide prevents the dissemination of HIV to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent HIV neuroinvasion.
... В их составе присутствуют трансмембранные и цитоплазматические белки. Трансмебранные белковые молекулы представлены окклюдином и клаудинами, которые ограничивают парацеллюлярный транспорт растворенных веществ и ионов, что создает высокое электрическое сопротивление барьера (более 1000 Ом /см) [41][42][43][44][45]. При исследовании ткани головного мозга методом Вестерн-блоттинга была показана экспрессия клаудинов-1, -3, -5 и -12, обеспечивающих повышение барьерных свойств эндотелия, а также клаудинов -2 и -17, образующих межклеточные поры для транспорта ионов и воды [43,[45][46][47]. ...
Abstract
The review addresses current ideas about the morphological and histophysiological features of the brain capillary endothelium with a special focus on the ultrastructure of endotheliocytes, which are part of the neurovascular unit. The relationship between their main characteristics and the implementation of the barrier function is discussed. The specificity of intercellular contacts in capillary endotheliocytes within the blood–brain barrier (BBB) and the structure of their cytoskeleton and glycocalyx are analyzed. The structural peculiarities of capillaries in the neurogenic niches, as well as in the anatomical formations, where the BBB is absent or poorly expressed, are considered. A separate section of the review is devoted to transcellular transport of substances (transcytosis) in cerebral endotheliocytes. Despite a wealth of publications on the subject, many issues remain unresolved, and the study of the structure and functioning of the cerebral microvascular endothelium is still on the agenda.
... ZO proteins anchor the tight junction complex to the actin cytoskeleton, making them essential for the assembly of claudins, occludins, and JAM-A. 28,29 Other examples of scaffolding proteins implicated in this protein network are symplekin, cingulin, 7H6, ZONAB (ZO-1associated nucleic acid-binding protein), AF-6 (ALL1-fused gene from chromosome 6 protein), and others (see Fig. 126.3). 12,20,25 Additionally, regulatory molecules such as G proteins (Gαi, RGS5) have been localized to the tight junction structure and play a major role in the regulation of BBB permeability. ...
... Given that this extracellular region, anchored by flanking transmembrane domains, must always be in close proximity to a membrane surface we suggest that the high propensity for helical structure we observe here may represent the native fold. High helical propensities in the extracellular loops of other integral membrane proteins including G-protein-coupled receptors 59,60 , transporters 61 , Na/ Ca exchangers 62 , tetraspanins 63 , and the cell division protein FtsX 64 have been reported previously. The presence of these helical elements is often thought to direct specific interactions with binding partners. ...
The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanSSC), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanSSC, and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. The VanSSC-binding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics.
... Les protéines suivantes composant les jonctions serrées sont les occludines, elles sont les premières protéines transmembranaires de ce complexe à avoir été décrites. Ces protéines sont responsables de l'interaction entre les protéines d'échafaudage (scaffold protein) et le cytosquelette d'actine (Li et al., 2005;Nusrat et al., 2005). Les occludines permettent aussi de réguler les propriétés d'adhésion entre les cellules. ...
La démence est caractérisée par une détérioration progressive des capacités mentales qui compromettent l’autonomie des patients. Le vieillissement en est le facteur de risque majeur. La population mondiale vieillissante, l’absence de traitement, ainsi que l’augmentation des individus touchés estimé à 50 millions, permettent d’estimer un coût de santé publique triplé à 4 trillions de dollars d’ici à 2050. Les études récentes tendent à montrer un rôle crucial de la dysfonction vasculaire dans les pathologies de démence. En effet une large étude neuropathologique basée sur l’autopsie des patients a permis de mettre en évidence que 80% des patients diagnostiqués de Maladie d’Alzheimer (MA) présentaient une pathologie vasculaire. Néanmoins, les mécanismes cellulaires et moléculaires entrainants ces dysfonctions vasculaires et altérant l’intégrité de la barrière hémato-encéphalique (BHE) dans le cas de la démence en général restent encore aujourd’hui méconnus. Au niveau moléculaire, la voie Wnt est impliqué dans le maintien de l’intégrité de la BHE. Le laboratoire a récemment identifié une E3-ubiquitine-ligase, PDZRN3 qui agit comme un médiateur de la signalisation Wnt non-canonique dans les cellules endothéliales en réprimant la voie Wnt canonique. Cette ubiquitine-ligase est impliquée dans la perméabilité vasculaire, faisant de cette dernière une cible pour réguler l’intégrité de cette barrière. Nous avons alors posé l’hypothèse suivante : La BHE ést déstabilisée dans la mise en place des démences dont la MA ; en maintenant l’intégrité de la BHE en réprimant la signalisation Wnt, permettrait de ralentir la sévérité de la MA et des altérations cognitives associées. Nous avons généré une lignée murine délétée pour Pdzrn3 dans l’endothélium de façon inductible (iECKO) et les avons soumises à une hypoperfusion cérébrale graduelle par la pose de constricteurs sur les artères carotides communes. Alors que les performances des souris contrôles sont impactées par l’hypoperfusion, celles des souris iECKO sont maintenues. Nos résultats montrent que les souris iECKO présentent une réduction significative des lésions cérébrales (micro-infarcts) ainsi qu’une réduction significative de la perte neuronale dans la zone CA1 de l’hippocampe jouant un rôle clé dans les processus mnésiques de reconnaisance spatiale. À l’inverse les souris surexprimant Pdzrn3 dans l’endothélium (iECOE) présentent le phénotype opposé à celui des souris iECKO, avec une détérioration mnésique plus importante que les contrôles associés à des fuites de la BHE, des lésions cérébrales, une perte neuronale hippocampale et une inflammation exacerbée. Nous avons aussi voulu étudier l’impact de la délétion dans les CE de Pdzrn3 dans le cadre de la MA. Nous avons généré et validé un modèle murin triple mutant en croisant les souris APP/PS1, modèle de MA, avec nos souris iECKO pour générer une souche APP/PS1 ; iECKO. Étonnamment, nos résultats montrent une réduction significative des dépôts amyloïdes à 6 mois dans les souris APP/PS1 ; Pdzrn3 iECKO comparés à leurs contrôles APP/PS1. À 8 mois les souris APP/PS1 apparaissent comme ayant une fonction cognitive sévèrement touchée dans un test mnésique de reconnaissance spatiale alors que les souris APP/PS1 ; Pdzrn3 iECKO ont une performance comparable à celle des contrôles de même âge non déments.
... E. histolytica expresses an occludin-like protein (55 kDa) with similarities to the extracellular loops of human occludin [87], which interacts in an homotypic manner to create and maintain the epithelial barrier [93]. The occludin-like protein has not been detected in the plasma membrane, but it is involved in the TEER dropping of T84 human colorectal carcinoma cells. ...
The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleria fowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.
... Occludin was one of the first tight junction transmembrane proteins described. Structurally, occludin contains two equal extracellular loops, four transmembrane domains and three cytoplasmic domains: one intracellular short turn, a small N-terminal domain and a long carboxyl (C-) terminal (Furuse et al. 1993;Li et al. 2005;Nusrat et al. 2005). Interestingly, the tight junction protein occludin is targeted during meningococcal infection: in vitro assays showed that meningococcal infection of brain endothelial cells resulted in proteolytic cleavage of occludin (Schubert-Unkmeir et al. 2010). ...
Neisseria meningitidis is one of the most common aetiological agents of bacterial meningitis, affecting predominantly children and young adults. The interaction of N. meningitidis with human endothelial cells lining blood vessels of the blood-cerebrospinal-fluid barrier (B-CSFB) is critical for meningitis development. In recent decades, there has been a significant increase in understanding of the molecular mechanisms involved in the interaction of N. meningitidis with brain vascular cells. In this review, we will describe how N. meningitidis adheres to the brain vasculature, may enter inside these cells, hijack receptor signalling pathways and alter host-cell responses in order to traverse the B-CSFB.
... A different claudin 4 peptide mimicking the second extracellular loop decreased CPE toxicity by protecting intestinal epithelial cells from the cytotoxic effects of full length CPE [78]. In contrast to the claudins, the beneficial effects of peptides mimicking occludin extracellular domains is less consistent and established [79][80][81][82][83]. ...
The intestinal epithelium serves as an important barrier between luminal contents and underlying tissue compartments. Barrier properties are established and maintained by the multiprotein complexes in the tight junction (TJ), adherens junction (AJ) and desmosomes (DM). Compromised intestinal epithelial barrier function resulting from changes in junctional protein expression and organization is associated with exposure to luminal antigens that result in mucosal inflammation, which in turn further exacerbates the epithelial barrier compromise. In this review we discuss regulatory mechanisms by which intercellular junction proteins are influenced by inflammatory mediators to influence intestinal epithelial barrier function and address potential therapeutic strategies to strengthen the barrier and restore mucosal homeostasis.
... However, other studies have reported that occludin expression can influence paracellular flux, which requires its C-terminal cytoplasmic domain. [44][45][46][47][48][49][50] Analogous to occludin, co-expression of MARVEL (membrane-associating) domain containing 3 (MARVELD3) with claudin 1 increases the number of TJ freeze-fracture strands. 51 MARVELD3 has been shown to affect cell behavior and survival by coupling TJs to MEKK1-JNK signaling. ...
The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states such as inflammatory bowel disease are associated with a leaky epithelial barrier resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory micro environment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation.
