Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability

Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.Under normal physiological conditions the blood-brain barrier and blood-retinal barrier regulate the transport of water, ions, amino acids, and waste products, between the neural parenchyma and blood (1). A high degree of well developed tight junctions (TJs)² in the vascular endothelium, in association with adherens junctions, contribute to both the blood-brain and blood-retinal barriers (2). Accumulating evidence suggests that a number of pathological eye diseases such as diabetes, retinopathy of prematurity, age-related macular degeneration, inflammation, and infectious diseases disrupt the TJs altering the blood-retinal barrier. Common mediators of vascular permeability and TJ deregulation are growth factors and cytokines that may induce macular edema and lead to loss of vision (1). Vascular endothelial growth factor (VEGF), in particular, induces vascular permeability and stimulates angiogenesis, contributing to disease pathogenesis in diabetic retinopathy and retinopathy of prematurity (3). VEGF also contributes to blood-brain barrier disruption with subsequent edema and angiogenesis in brain tumors and stroke (4). Recent advances in biomedical research have provided therapeutic approaches to neutralize VEGF; however, these strategies have not yet demonstrated effective resolution of diabetic macular edema (5, 6).TJs control the paracellular flux of solutes and fluids across the blood-brain and blood-retinal barriers. Several transmembrane proteins including occludin, tricellulin, the claudin family, and junction adhesion molecules are thought to confer adhesion to the TJ barrier and to be organized by members of the zonula occludens family (ZO-1, -2, or -3) (7–9). Experimental evidence has established that the claudins confer barrier properties and claudin-5 specifically contributes to the vascular component of the blood-brain barrier demonstrated by gene deletion studies (10). In contrast, the function of occludin in paracellular flux has remained less clear. Mice with occludin gene deletion continue to form TJs in gut epithelia with normal barrier properties (11). However, studies have also demonstrated that diabetes reduces occludin content in rat retina (12) and alters its distribution from continuous cell border localization to intracellular puncta (13). These observations suggest that the intracellular trafficking of TJ proteins promotes paracellular flux and vascular permeability in diabetic animals (12, 14).VEGF was originally identified as a vascular permeability factor as well as a pro-angiogenic growth factor (15, 16). Both biological effects exacerbate the pathology of retinal vascular diseases (17), and they are mediated via intracellular signal transduction, especially based on the phosphorylation of Src, protein kinase C, and so on (18). Additionally, VEGF treatment and diabetes induce occludin phosphorylation in rat retinal vasculature and endothelial cell culture coincident with increased permeability (19). Recently, using mass spectrometry five occludin phosphorylation sites were identified in retinal endothelial cell culture after VEGF treatment (20). Among these sites, phosphorylation at Ser-490 was shown to increase in response to VEGF treatment. However, no evidence has directly demonstrated the contribution of occludin phosphorylation to VEGF-induced endothelial permeability or defined the mechanism by which phosphorylation of occludin alters paracellular flux.Modification of proteins with monomeric or polymeric ubiquitin chains contributes to control of multiple biological functions including protein degradation, intracellular trafficking, translational regulation, and DNA repair (21). Phosphorylation of receptor tyrosine kinases, such as epidermal growth factor receptor or vascular endothelial growth factor receptor-2, is followed by ubiquitination and regulated trafficking to endosomes. This endocytosis process depends on the interaction between the ubiquitinated receptors and carrier proteins that possess a ubiquitin interacting motif (UIM) such as Epsin, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (21–24). Recent publications have demonstrated that occludin can be ubiquitinated targeting the protein for degradation through the ubiquitin-proteasome system in epithelial cell types (25, 26). Here we demonstrate that phosphorylation of occludin at Ser-490 is necessary for occludin ubiquitination in response to VEGF in endothelial cells. Furthermore, the ubiquitination promotes interaction of occludin with UIM containing modulators of trafficking and regulates the internalization of TJ proteins altering endothelial permeability. Together, these results suggest that occludin phosphorylation and subsequent ubiquitination are necessary for VEGF-induced TJ trafficking and endothelial permeability.     

Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116–319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10–13, 15, 16, 18–20, and 22. Substitution peptide mapping identified the central motif ¹⁴⁸NPL¹⁵⁰VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn¹⁴⁸ and Leu¹⁵⁰ of full-length claudin-3 in the binding. CPE-(116–319) and CPE-(194–319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116–319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116–319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116–319) and CPE-(194–319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.The clinical use of many promising drug candidates is impeded by unacceptable pharmacokinetics (1). The ability of a drug to pass through tissue barriers is a major determinant for its delivery. In epithelia and endothelia, the paracellular route is blocked by tight junctions (TJ).⁴ Different approaches have been used to enhance transcellular drug delivery. These include the use of influx transporters, blocking of efflux transporters, or receptor-mediated endocytosis (2). Alternative approaches aim to enhance paracellular permeation of drugs by loosening the TJ (3, 4). This strategy has the advantage that it could improve the delivery of structurally unrelated drugs, and the drug itself does not have to be modified. Although different TJ modulators have been described, most of these are based on surfactants or chelators (3). These often have low tissue specificity and cause severe side effects, e.g. exfoliation of cells, which irreversibly compromise the barrier functions (5, 6). Fewer side effects may be obtained by more specific modulation of a molecular key component of the TJ (7).TJ consist of transmembrane proteins, mainly the tetraspan proteins of the claudin family, as well as occludin and tricellulin (8). Other molecules associated with TJ include membrane-bound scaffolding and signaling proteins (9). However, claudins (Cld) are the major functional constituent of TJ (10). Claudins tighten the paracellular space, selectively for tissue, size, and charge. The tissue-specific combination of the claudin subtypes present in heteropolymers is assumed to determine the permeability properties of TJ (11). It was therefore proposed that tissue-specific drug delivery via the paracellular route would be possible by modulation of the barrier-function of claudins in a subtype-specific manner (7).A subset of claudins, e.g. Cld3 and -4 but not -1 and -2, have been shown to be receptors for Clostridium perfringens enterotoxin (CPE) with high association constants of about 10⁸ m⁻¹ (12). CPE causes one of the most common food-borne diseases (13). It consists of two functional domains, an N-terminal region that mediates the cytotoxic effect and the C-terminal region (CPE-(184–319)), which binds to extracellular loop 2 (ECL2) of Cld3 but not of Cld1 nor to the ECL1 of Cld3 (12). Treatment of epithelial monolayers with non-cytotoxic CPE-(184–319) increases paracellular permeability (14). CPE-(184–319) enhanced drug absorption in rat jejunum 400-fold relative to sodium caprate, which is in clinical use (15). Thus, CPE is a promising tool to specifically modulate claudins, the key constituents of TJ, and thereby to enhance paracellular drug delivery. In addition, some studies have suggested the use of CPE for the chemotherapy of tumors overexpressing claudins (16–18).Cld1 and -5 are potential targets for transepidermal and brain drug delivery, respectively (19, 20). However, it has been reported that these claudins do not interact with CPE (12). Modification of CPE could enhance and/or shift its claudin-subtype specificity. Therefore, the design of CPE-based TJ modulators could permit efficient claudin subtype-specific modulation, which would also be tissue-specific modulation of TJ. To achieve this, an understanding of the molecular mechanism of the CPE-claudin interaction is a necessary prerequisite. In this study, we identify the residues within the ECL2 of Cld3 that are involved in interaction with CPE.     

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin^ΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.Tight junctions seal the paracellular space in simple epithelia, such as those lining the lungs, intestines, and kidneys (Anderson et al., 2004 ; Fanning and Anderson, 2009 ; Shen et al., 2011 ). In the intestine, reduced paracellular barrier function is associated with disorders in which increased paracellular flux of ions and molecules contributes to symptoms such as diarrhea, malabsorption, and intestinal protein loss. Recombinant tumor necrosis factor (TNF) can be used to model this barrier loss in vitro or in vivo (Taylor et al., 1998 ; Clayburgh et al., 2006 ), and TNF neutralization is associated with restoration of intestinal barrier function in Crohn''s disease (Suenaert et al., 2002 ). Further, in vivo and in vitro studies of intestinal epithelia show that TNF-induced barrier loss requires myosin light chain kinase (MLCK) activation (Zolotarevsky et al., 2002 ; Clayburgh et al., 2005 , 2006 ; Ma et al., 2005 ; Wang et al., 2005 ). The resulting myosin II regulatory light chain (MLC) phosphorylation drives occludin internalization, which is required for cytokine-induced intestinal epithelial barrier loss (Clayburgh et al., 2005 , 2006 ; Schwarz et al., 2007 ; Marchiando et al., 2010 ). In addition, transgenic EGFP-occludin expression in vivo limits TNF-induced depletion of tight junction–associated occludin, barrier loss, and diarrhea (Marchiando et al., 2010 ). Conversely, in vitro studies show that occludin knockdown limits TNF-induced barrier regulation (Van Itallie et al., 2010 ). The basis for this discrepancy is not understood.One challenge is that, despite being identified 20 yr ago (Furuse et al., 1993 ), the contribution of occludin to tight junction regulation remains incompletely defined. The observation that occludin-knockout mice are able to form paracellular barriers and do not have obvious defects in epidermal, respiratory, or bladder tight junction function (Saitou et al., 2000 ; Schulzke et al., 2005 ) led many to conclude that occludin is not essential for tight junction barrier function. It is important to note, however, that barrier regulation in response to stress has not been studied in occludin-deficient animals.We recently showed that dephosphorylation of occludin serine-408 promotes assembly of a complex composed of occludin, ZO-1, and claudin-2 that inhibits flux across size- and charge-selective channels termed the pore pathway (Anderson and Van Itallie, 2009 ; Turner, 2009 ; Raleigh et al., 2011 ; Shen et al., 2011 ). Although this demonstrates that occludin can serve a regulatory role, it does not explain the role of occludin in TNF-induced barrier loss, which increases flux across the size- and charge-nonselective leak pathway (Wang et al., 2005 ; Weber et al., 2010 ). In vitro studies, however, do suggest that occludin contributes to leak pathway regulation, as occludin knockdown in either Madin–Darby canine kidney (MDCK) or human intestinal (Caco-2) epithelial monolayers enhances leak pathway permeability (Yu et al., 2005 ; Al-Sadi et al., 2011 ; Ye et al., 2011 ). Taken as a whole, these data suggest that occludin organizes the tight junction to limit leak pathway flux, whereas occludin removal, either by knockdown or endocytosis, enhances leak pathway flux.To define the mechanisms by which occludin regulates the leak pathway, we analyzed the contributions of occludin, as well as specific occludin domains, to basal and TNF-induced barrier regulation. The data indicate that TNF destabilizes tight junction–associated occludin via interactions mediated by the C-terminal coiled-coil occludin/ELL domain (OCEL). Further, these OCEL-mediated events are required for TNF-induced barrier regulation. Thus these data provide new insight into the structural elements and mechanisms by which occludin regulates leak pathway paracellular flux.     

The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection

The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.Hepatitis C virus (HCV) is a small enveloped positive-strand RNA virus that belongs to the Flaviviridae family (20). More than 80% of acute infections become chronic, which eventually progress to cirrhosis and hepatocellular carcinoma (28). HCV infects mainly hepatocytes, but the precise mechanisms of infection are largely unknown (11). The HCV particle consists of a nucleocapsid surrounded by a lipid bilayer in which the two envelope glycoproteins (HCVgp), E1 and E2, are anchored as a heterodimer and play a major role in HCV entry (20). The development of an infectious cell culture model based on the production of infective HCV particles (cell culture-derived HCV particles [HCVcc]) (34) and the generation of HCV pseudotyped retroviral particles (HCVpp) (4) have provided powerful tools to study the HCV cycle. HCV entry is a complex multistep process that requires the presence of several factors. There are multiple pieces of evidence for the involvement of host cell proteins in HCV entry, including glycosaminoglycans, the low-density lipoprotein receptor, scavenger receptor class B type I (SR-BI), and the tetraspanin CD81 (11). Recently, claudin-1, a tight junction (TJ) component, has been identified as a coreceptor required for a late step in HCV entry (13).TJs are major components of cell-cell adhesion complexes and are composed of integral membrane proteins, including occludin and claudins, which associate with actin cytoskeleton-interacting proteins, such as zonula occludens protein 1 (ZO-1) (2). These structures maintain cell polarity, separating apical from basolateral membrane domains, and form a paracellular barrier that allows the selective passage of certain solutes (2). In hepatocytes, TJs seal the bile canaliculi and form the intercellular barrier between bile and blood (12). Recently, we have shown that TJ-associated proteins occludin and claudin-1 disappeared from their normal localization in both HCV-infected and genomic HCV replicon-containing Huh7 cells. Furthermore, TJ function was also altered in these cells (5). In this matter, we have reported an intracellular interaction between E2 and occludin (5). Moreover, it has been reported that claudin-1 and several TJ-associated proteins, such as coxsackievirus and adenovirus receptor (35) and junctional adhesion molecule (JAM) (3), act as virus (co)receptors. Since coxsackievirus entry across epithelial TJs requires occludin (10), we have explored the role of occludin in HCV infection.     

Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis

Claudins form size- and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp⁶⁵ lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri)benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile⁶⁶ is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr³⁵ is located at the protein surface outside of the pore.Sheets of polarized epithelia constitute barriers that separate fluid compartments of different chemical composition and mediate exchange of solutes and ions via transcellular and paracellular pathways. A large body of evidence suggests that transport via the paracellular pathway occurs through pores in the tight junctions that are formed by tetraspan membrane proteins, known as claudins (1–3).Our current understanding of paracellular pores is that they are size- and charge-selective water-filled channels that, in contrast to channels for transmembrane transport, are oriented parallel instead of perpendicular to the lipid layer of the cell membrane. Size exclusion experiments suggest a pore diameter of 6.4–8 Å (4, 5). Furthermore, site-directed mutagenesis and overexpression of claudins in epithelial cells identified the first extracellular domain as playing an important role in the charge selectivity of paracellular transport (6–8). The first extracellular domain of claudins contains various basic and acidic amino acids, some of which are conserved in different claudin isoforms, and these could be involved in the mechanism of ion permeation. Several studies have demonstrated homo- or heterotypic interaction of claudins, suggesting that paracellular pores are formed by oligomers of claudins (9–11). Taken together, significant progress has been made in uncovering the nature of the paracellular pathway and mechanisms of selectivity of paracellular ion permeation. However, it is unknown how the extracellular domains of claudins fold to form paracellular pores and which amino acid residues line the pathway of ion diffusion.Epithelia in vivo and epithelial cell lines express characteristic sets of different claudin isoforms that determine paracellular permeability and permselectivity. Claudin-2 is expressed in epithelia with a high capacity for passive paracellular cation transport, such as the epithelium lining the proximal renal tubules (12). The transfection of claudin-2 into high resistance Madin-Darby canine kidney (MDCK)² type I cells converts the tight junction from a “tight” into a “leaky” paracellular barrier by selectively increasing Na⁺ permeability (13, 14), suggesting a physiologic role of claudin-2 in creating paracellular Na⁺ channels. Because of the high signal/noise ratio of the claudin-2-induced permeability, this isoform provides an excellent model to study paracellular transport. We have recently generated a stable expression system of claudin-2 in MDCK I cells under the control of a TetOff promoter. This inducible system allows us to specifically determine the macroscopic conductance and permeability of claudin-2 pores by subtracting background measurements of uninduced cells. Using this expression system, we could recently demonstrate that the cation selectivity of claudin-2 cells is mediated by electrostatic interaction of partially dehydrated permeating cations with aspartate 65 (5). However, further investigations are necessary to study the position and function of this and other residues of the first extracellular domain and to elucidate their role in the transport mechanism of paracellular pores.The substituted cysteine accessibility method (SCAM), developed by the Karlin group, has proved to be a powerful tool in the mapping of the structures of membrane ion channels and transport proteins (15, 16). In SCAM, thiol-reactive reagents are used to covalently modify endogenous cysteines, or cysteines introduced into a protein by site-directed mutagenesis. SCAM can be used to study channel-lining amino acid side chains, the secondary structures of membrane-spanning segments, and the localization of selectivity filters, channel gates, and inhibitor binding sites.Here, we used SCAM to analyze the paracellular pathway of ion permeation across claudin-2 transfected MDCK I cells. Our data show that thiol-reactive reagents strongly block ion transport in at least one of the cysteine mutants that we have generated and, thus, provide a tool to map residues that line the paracellular pore.     

The Regulation of Vascular Endothelial Growth Factor-induced Microvascular Permeability Requires Rac and Reactive Oxygen Species

Vascular permeability is a complex process involving the coordinated regulation of multiple signaling pathways in the endothelial cell. It has long been documented that vascular endothelial growth factor (VEGF) greatly enhances microvascular permeability; however, the molecular mechanisms controlling VEGF-induced permeability remain unknown. Treatment of microvascular endothelial cells with VEGF led to an increase in reactive oxygen species (ROS) production. ROS are required for VEGF-induced permeability as treatment with the free radical scavenger, N-acetylcysteine, inhibited this effect. Additionally, treatment with VEGF caused ROS-dependent tyrosine phosphorylation of both vascular-endothelial (VE)-cadherin and β-catenin. Rac1 was required for the VEGF-induced increase in permeability and adherens junction protein phosphorylation. Knockdown of Rac1 inhibited VEGF-induced ROS production consistent with Rac lying upstream of ROS in this pathway. Collectively, these data suggest that VEGF leads to a Rac-mediated generation of ROS, which, in turn, elevates the tyrosine phosphorylation of VE-cadherin and β-catenin, ultimately regulating adherens junction integrity.Endothelial cells line the inside of blood vessels and serve as a barrier between circulating blood and the surrounding tissues. Endothelial permeability is mediated by two pathways: the transcellular pathway and the paracellular pathway. In the transcellular pathway material passes through the cells, whereas in the paracellular pathway fluid and macromolecules pass between the cells. The paracellular pathway is regulated by the properties of endothelial cell-cell junctions (1–3). Changes in the permeability of this barrier are tightly regulated under normal physiological conditions. However, dysregulated vascular permeability is observed in many life-threatening conditions, including heart disease, cancer, stroke, and diabetes.VEGF² was first discovered as a potent vascular permeability factor that stimulated a rapid and reversible increase in microvascular permeability without damaging the endothelial cell (4, 5). VEGF was later shown to be a selective growth factor for endothelial cells, capable of promoting migration, growth, and survival (6). Considerable progress has been made toward understanding the signaling events by which VEGF promotes growth and survival (7). However, the mechanism through which VEGF promotes microvascular permeability remains incompletely understood.VE-cadherin is an endothelial cell-specific adhesion molecule that connects adjacent endothelial cells (8, 9). While the barrier function of the endothelium is supported by multiple cell-cell adhesion systems, disruption of VE-cadherin is sufficient to disrupt intercellular junctions (9–11). Earlier studies have demonstrated increased permeability both in vitro and in vivo after treatment with VE-cadherin-blocking antibodies (9, 12). Additionally, VE-cadherin is required to prevent disassembly of blood vessel walls (11, 13) and to coordinate the passage of macromolecules through the endothelium (14, 15). Tyrosine phosphorylation may provide the regulatory link, as increased phosphorylation of cadherins and potential dissociation of the cadherin/catenin complex results in decreased cell-cell adhesion and increased permeability (16, 17).Recent evidence has demonstrated that Rac1-induced reactive oxygen species (ROS) disrupt VE-cadherin based cell-cell adhesion (18). The mechanisms by which ROS affect endothelial permeability have not been fully characterized. VEGF has been reported to induce NADPH oxidase activity and induce the formation of ROS (19, 20). A direct link between Rac and ROS in a non-phagocytic cell was shown in 1996, when it was demonstrated that activated Rac1 resulted in the increased generation of ROS in fibroblasts (21). Several studies have subsequently implicated Rac-mediated production of ROS in a variety of cellular responses, in particular in endothelial cells (22, 23). These data suggest that ROS may play a critical role in integrating signals from VEGF and Rac to regulate the phosphorylation of VE-cadherin and ultimately the integrity of the endothelial barrier.In the present study we sought to determine the mechanism by which VEGF regulates microvascular permeability. Our results show that VEGF treatment of human microvascular endothelial cells results in the Rac-dependent production of ROS and the subsequent tyrosine phosphorylation of VE-cadherin and β-catenin. The phosphorylation of VE-cadherin and β-catenin are dependent on Rac and ROS and result in decreased junctional integrity and enhanced vascular permeability.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]