Cingulin binds to the ZU5 domain of scaffolding protein ZO-1 to promote its extended conformation, stabilization, and tight junction accumulation.

Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known. Here, we used glutathione-S-transferase pulldowns and immunofluorescence microscopy to show that CGN and CGNL1 bind to the C-terminal ZU5 domain of ZO-1 and that this domain is required for CGN and CGNL1 recruitment to TJs and to phase-separated ZO-1 condensates in cells. We show that KO of CGN, but not CGNL1, results in decreased accumulation of ZO-1 at TJs. Furthermore, ZO-1 lacking the ZU5 domain showed decreased accumulation at TJs, was detectable along lateral contacts, had a higher mobile fraction than full-length ZO-1 by fluorescence recovery after photobleaching analysis, and had a folded conformation, as determined by structured illumination microscopy of its N-terminal and C-terminal ends. The CGN-ZU5 interaction promotes the extended conformation of ZO-1, since binding of the CGN-ZO-1 interaction motif region to ZO-1 resulted in its interaction with DbpA in cells and in vitro. Together, these results show that binding of CGN to the ZU5 domain of ZO-1 promotes ZO-1 stabilization and accumulation at TJs by promoting its extended conformation.

Down-regulation of diesel particulate matter-induced airway inflammation by the PDZ motif peptide of ZO-1.

Although diesel airborne particulate matter (PM2.5) has been known to play a role in many human diseases, there is no direct evidence that therapeutic drugs or proteins can diminish PM2.5-induced diseases. Nevertheless, studies examining the negative control mechanisms of PM2.5-induced diseases are critical to develop novel therapeutic medications. In this study, the consensus PDZ peptide of ZO-1 inhibited PM2.5-induced inflammatory cell infiltration, pro-inflammatory cytokine gene expression, and TEER in bronchoalveolar lavage (BAL) fluid and AM cells. Our data indicated that the PDZ domain in ZO-1 is critical for regulation of the PM2.5-induced inflammatory microenvironment. Therefore, the PDZ peptide may be a potential therapeutic candidate during PM-induced respiratory diseases.

Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.

Spatial Overlap of Claudin- and Phosphatidylinositol Phosphate-Binding Sites on the First PDZ Domain of Zonula Occludens 1 Studied by NMR.

Background: The tight junction is an intercellular adhesion complex composed of claudins (CLDs), occludin, and the scaffolding proteins zonula occludens 1 (ZO-1) and its two paralogs ZO-2 and ZO-3. ZO-1 is a multifunctional protein that contains three PSD95/Discs large/ZO-1(PDZ) domains. A key functional domain of ZO-1 is the first PDZ domain (ZO-1(PDZ1)) that recognizes the conserved C-termini of CLDs. Methods: In this study, we confirmed that phosphoinositides bound directly to ZO-1(PDZ1) by biochemical and solution NMR experiments. We further determined the solution structure of mouse ZO-1(PDZ1) by NMR and mapped the phosphoinositide binding site onto its molecular surface. Results: The phosphoinositide binding site was spatially overlapped with the CLD-binding site of ZO-1(PDZ1). Accordingly, inositol-hexaphosphate (phytic acid), an analog of the phosphoinositide head group, competed with ZO-1(PDZ)-CLD interaction. Conclusions: The results suggested that the PDZ domain⁻phosphoinositide interaction plays a regulatory role in biogenesis and homeostasis of the tight junction.

Targeting Neph1 and ZO-1 protein-protein interaction in podocytes prevents podocyte injury and preserves glomerular filtration function.

Targeting protein-protein interaction (PPI) is rapidly becoming an attractive alternative for drug development. While drug development commonly involves inhibiting a PPI, in this study, we show that stabilizing PPI may also be therapeutically beneficial. Junctional proteins Neph1 and ZO-1 and their interaction is an important determinant of the structural integrity of slit diaphragm, which is a critical component of kidney's filtration system. Since injury induces loss of this interaction, we hypothesized that strengthening this interaction may protect kidney's filtration barrier and preserve kidney function. In this study, Neph1-ZO-1 structural complex was screened for the presence of small druggable pockets formed from contributions from both proteins. One such pocket was identified and screened using a small molecule library. Isodesmosine (ISD) a rare naturally occurring amino acid and a biomarker for pulmonary arterial hypertension was selected as the best candidate and to establish the proof of concept, its ability to enhance Neph1-CD and ZO-1 binding was tested. Results from biochemical binding analysis showed that ISD enhanced Neph1 and ZO-1 interaction under in vitro and in vivo conditions. Importantly, ISD treated podocytes were resistant to injury-induced loss of transepithelial permeability. Finally, mouse and zebrafish studies show that ISD protects from injury-induced renal damage.

Acute ocular hypertension disrupts barrier integrity and pump function in rat corneal endothelial cells.

Acute ocular hypertension (AOH) frequently compromises corneal endothelial cell (CEC) function in clinical practice. This type of stress induces corneal oedema and a decrease in the corneal endothelial cell density (ECD). The anterior chamber of the right eye of Sprague-Dawley rats was irrigated with Balanced Salt Solution (BSS) for two hours, and the left eye served as a control to determine the time-dependent effects of AOH on endothelial cell morphology and function. The average intraocular pressure (IOP) increased to 82.6 ± 2.3 mmHg (normal range: 10.2 ± 0.4 mmHg) during anterior irrigation. Very soon after initiating irrigation, corneal oedema became evident and the cornea exhibited a significant increase in permeability to FITC-dextran. The peripheral ECD was significantly reduced, and the morphology of CECs became irregular and multiform. The structures of the zonula occludens-1 (ZO-1) and F-actin were severely disrupted. In addtion, Na,K-ATPase exhibited a dispersed expression pattern. Two days after irrigation, obvious CEC proliferation was observed, the ECD recovered to a normal level, and F-actin was dispersed throughout the cytoplasm. Seven days later, the CEC structure and function were nearly normalized. Based on the results obtained using this model, an acute IOP crisis exerts transient deleterious effects on CEC structure and function in rats.

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture.

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc.

[High frequency of ancestral allele of the TJP1 polymorphism rs2291166 in Mexican population, conformational effect and applications in surgery and medicine]. / Alta frecuencia del alelo ancestral del polimorfismo rs2291166 de TJP1 en población mexicana, efecto conformacional así como las aplicaciones en cirugía y medicina.

BACKGROUND: TJP1 gene encodes a ZO-1 protein that is required for the recruitment of occludins and claudins in tight junction, and is involved in cell polarisation. It has different variations, the frequency of which has been studied in different populations. In Mexico there are no studies of this gene. These are required because their polymorphisms can be used in studies associated with medicine and surgery. Therefore, the aim of this study was to estimate the frequency of alleles and genotypes of rs2291166 gene polymorphism TJP1 in Mexico Mestizos population, and to estimate the conformational effect of an amino acid change. MATERIAL AND METHODS: A total of 473 individuals were included. The rs2291166 polymorphism was identified PASA PCR-7% PAGE, and stained with silver nitrate. The conformational effect of amino acid change was performed in silico, and was carried out with servers ProtPraram Tool and Search Database with Fasta. RESULTS: The most frequent allele in the two populations is the ancestral allele (T). A genotype distribution similar to other populations was found. The polymorphism is in Hardy-Weinberg, p>0.05. Changing aspartate to alanine produced a conformational change. CONCLUSIONS: The study reveals a high frequency of the ancestral allele at rs2291166 polymorphism in the Mexican population.

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins.

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the -6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 ß2-ß3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.

Three-dimensional human arterial wall models for in vitro permeability assessment of drug and nanocarriers.

Monolayers of endothelial cells (1L-ECs) have been generally used as in vitro vascular wall models to study the vascular mechanisms and transport of substances. However, these two-dimensional (2D-) system cannot represent the properties of native vascular walls which have a 3D-structure and are composed of not only ECs, but also smooth muscle cells (SMCs) and other surrounding tissues. Here in, 5-layered (5L) 3D-arterial wall models (5L-AWMs) composed of EC monolayer and 4-layered SMCs were constructed by hierarchical cell manipulation. We applied the 5L-AWMs to evaluate their barrier function and permeability to nano-materials in order to analyze drug, or drug nanocarrier permeability to the blood vessel in vitro. Barrier property of the 3D-AWMs was confirmed by Zonula occludens (ZO-1) staining and their transendothelial electrical resistance (TEER), which was comparable to 1L-ECs, while the SMCs showed close to zero. The effect of substance size to permeability across the 5L-AWMs was clearly observed from dextrans with various molecular weights, which agreed well with the known phenomena of the in vivo blood vessels. Importantly, transport of nano-materials could be observed across the depth of 5L-AWMs, suggesting the advantage of 3D-AWMs over general 2D-systems. By using this system, we evaluate the transport of 35 nm phenylalanine-modified poly(γ-Glutamic Acid) nanoparticles (γ-PGA-Phe NPs) as a candidate of biodegradable drug carrier. Interestingly, despite of having comparable size to dextran-2000 k (28 nm), the γ-PGA-Phe NPs distinctly showed approximately 20 times faster transport across the 5L-AWMs, suggesting the effect of intrinsic properties of the substance on the transport. This in vitro evaluation system using the 3D-AWMs is therefore useful for the design and development of nano-drug carriers for treatment of vascular diseases, such as atherosclerosis.

A structural portrait of the PDZ domain family.

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended ß-strand conformation by interacting in an antiparallel fashion with a PDZ ß-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.

A bioengineered array of 3D microvessels for vascular permeability assay.

Blood vessels exhibit highly regulated barrier function allowing selective passage of macromolecules. Abnormal vascular permeability caused by disorder in barrier function is often associated with various pathological states such as tumor progression or pulmonary fibrosis. There are no realistic in vitro models for measuring vascular permeability as most models are limited to mimicking anatomical structural properties of in vivo vessel barriers. This paper presents a reliable microfluidic-based chip for measuring permeability by engineering tubular perfusable microvessels. This platform is compatible with high resolution, live-cell time-lapse imaging and high throughput permeability measurements. The microvessels were formed by natural angiogenic process and thus exhibit reliable barrier properties with permeability coefficient of 1.55×10(-6)cm/s (for 70kDa FITC-dextran). The bioengineered microvessels showed properties similar to in vivo vessels in terms of cell-cell junction expression (ZO-1, Claudin-5 and VE-cadherin) and response to agonists such as histamine and TNF-α. We showed that hyperpermeability of the tumor microvessel could be normalized with anti-VEGF (bevacizumab) treatment, consistent with the mechanism of action for bevacizumab. The method developed here provides a relatively simple, robust technique for assessing drug effects on permeability of microvessels with a number of potential applications in fundamental vascular biology as well as drug screening.

Sorting nexin 27 (SNX27) associates with zonula occludens-2 (ZO-2) and modulates the epithelial tight junction.

Proteins of the SNX (sorting nexin) superfamily are characterized by the presence of a PX (Phox homology) domain and associate with PtdIns3P (phosphatidylinositol-3-monophosphate)-rich regions of the endosomal system. SNX27 is the only sorting nexin that contains a PDZ domain. In the present study, we used a proteomic approach to identify a novel interaction between SNX27 and ZO-2 [zonula occludens-2; also known as TJP2 (tight junction protein 2)], a component of the epithelial tight junction. The SNX27-ZO-2 interaction requires the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2. When tight junctions were perturbed by chelation of extracellular Ca2+, ZO-2 transiently localized to SNX27-positive early endosomes. Depletion of SNX27 in mpkCCD (mouse primary kidney cortical collecting duct) cell monolayers resulted in a decrease in the rate of ZO-2, but not ZO-1, mobility at cell-cell contact regions after photobleaching and an increase in junctional permeability to large solutes. The findings of the present study identify an important new SNX27-binding partner and suggest a role for endocytic pathways in the intracellular trafficking of ZO-2 and possibly other tight junction proteins. Our results also indicate a role for SNX27-ZO-2 interactions in tight junction maintenance and function.

Phosphoinositides and PDZ domain scaffolds.

The discovery that PSD-95/Discs large/ZO-1 (PDZ) domains can function as lipid-binding modules, in particular interacting with phosphoinositides (PIs), was made more than 10 years ago (Mol Cell 9(6): 1215-1225, 2002). Confirmatory studies and a series of functional follow-ups established PDZ domains as dual specificity modules displaying both peptide and lipid binding, and prompted a rethinking of the mode of action of PDZ domains in the control of cell signaling. In this chapter, after introducing PDZ domains, PIs and methods for studying protein-lipid interactions, we focus on (i) the prevalence and the specificity of PDZ-PIs interactions, (ii) the molecular determinants of PDZ-PIs interactions, (iii) the integration of lipid and peptide binding by PDZ domains, (iv) the common features of PIs interacting PDZ domains and (v) the regulation and functional significance of PDZ-PIs interactions.

In vitro formation and characterization of a perfusable three-dimensional tubular capillary network in microfluidic devices.

This paper describes the in vitro formation and characterization of perfusable capillary networks made of human umbilical vein endothelial cells (HUVECs) in microfluidic devices (MFDs). Using this platform, an array of three-dimensional (3D) tubular capillaries of various dimensions (50-150 µm in diameter and 100-1600 µm in length) can be formed reproducibly. To generate connected blood vessels, MFDs were completely filled with fibrin gel and subsequently processed to selectively leave behind gel structures inside the bridge channels. Following gel solidification, HUVECs were coated along the gel walls, on opposite ends of the patterned 3D fibrin gel. After 3-4 days, HUVECs migrating into the fibrin gel from opposite ends fused with each other, spontaneously forming a connected vessel that expressed tight junction proteins (e.g., ZO-1), which are characteristic of post-capillary venules. With ready access to a perfusable capillary network, we demonstrated perfusion of the vessels and imaged red blood cells (RBCs) and beads flowing through them. The results were reproducible (∼50% successful perfusable capillaries), consistent, and could be performed in a parallel manner (9 devices per well plate). Additionally, compatibility with high resolution live-cell microscopy and the possibility of incorporating other cell types makes this a unique experimental platform for investigating basic and applied aspects of angiogenesis, anastomosis, and vascular biology.