scholarly journals Contribution of protein-protein interactions to the endothelial barrier-stabilizing function of KRIT1

2021 ◽  
Author(s):  
Harsha Swamy ◽  
Angela J. Glading
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Protein Interactions ◽  
Barrier Function ◽  
Protein Localization ◽  
Adherens Junction ◽  
Endothelial Barrier ◽  
Protein Protein Interactions ◽  
Cortical Actin ◽  
Ptb Domain

Krev-interaction trapped 1 (KRIT1) is an endothelial scaffold protein that promotes adherens junction (AJ) stability. The precise mechanism by which KRIT1 promotes barrier stabilization is unclear. We tested the ability of a panel of KRIT1 constructs containing mutations that inhibit Rap1 binding, ICAP1 binding, disrupt KRIT1's protein tyrosine binding domain (PTB), or direct KRIT1 to the plasma membrane, either alone or in combination, to restore barrier function in KRIT1-deficient endothelial cells. We found that ablating the 192NPAY195 motif or disrupting the PTB domain was sufficient to restore AJ protein localization and barrier function to control levels, irrespective of the junctional localization of KRIT1 or Rap1 binding. The ability of our KRIT1 constructs to rescue AJ/barrier function in KRIT1 depleted endothelial cells correlated with decreased 1 integrin activity and maintenance of cortical actin fibers. Together, our findings indicate that Rap1 binding, ICAP1 binding, and junctional localization are not required for the ability of KRIT1 to stabilize endothelial contacts, and suggest that the ability of KRIT1 to limit integrin activity may be involved in barrier stabilization.

scholarly journals Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium

2010 ◽  
Vol 298 (3) ◽  
pp. L361-L370 ◽  
Author(s):  
K. L. Grinnell ◽  
B. Casserly ◽  
E. O. Harrington
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Barrier Function ◽  
Adherens Junction ◽  
Fiber Formation ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Protein Tyrosine

Pulmonary edema is mediated in part by disruption of interendothelial cell contacts. Protein tyrosine phosphatases (PTP) have been shown to affect both cell-extracellular matrix and cell-cell junctions. The SH2 domain-containing nonreceptor PTP, SHP2, is involved in intercellular signaling through direct interaction with adherens junction proteins. In this study, we examined the role of SHP2 in pulmonary endothelial barrier function. Inhibition of SHP2 promoted edema formation in rat lungs and increased monolayer permeability in cultured lung endothelial cells. In addition, pulmonary endothelial cells demonstrated a decreased level of p190RhoGAP activity following inhibition of SHP2, events that were accompanied by a concomitant increase in RhoA activity. Furthermore, immunofluorescence microscopy confirmed enhanced actin stress fiber formation and diminished interendothelial staining of adherens junction complex-associated proteins upon SHP2 inhibition. Finally, immunoprecipitation and immunoblot analyses demonstrated increased tyrosine phosphorylation of VE-cadherin, β-catenin, and p190RhoGAP proteins, as well as decreased association between p120-catenin and VE-cadherin proteins. Our findings suggest that SHP2 supports basal pulmonary endothelial barrier function by coordinating the tyrosine phosphorylation profile of VE-cadherin, β-catenin, and p190RhoGAP and the activity of RhoA, signaling molecules important in adherens junction complex integrity.

scholarly journals De novo design of a reversible phosphorylation-dependent switch for membrane targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leon Harrington ◽  
Jordan M. Fletcher ◽  
Tamara Heermann ◽  
Derek N. Woolfson ◽  
Petra Schwille
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
De Novo ◽  
Protein Localization ◽  
Protein Structures ◽  
Spatiotemporal Pattern ◽  
Membrane Targeting ◽  
Protein Protein Interactions ◽  
Reversible Phosphorylation ◽  
Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

scholarly journals β-Catenin is a pH sensor with decreased stability at higher intracellular pH

2018 ◽  
Vol 217 (11) ◽  
pp. 3965-3976 ◽  
Author(s):  
Katharine A. White ◽  
Bree K. Grillo-Hill ◽  
Mario Esquivel ◽  
Jobelle Peralta ◽  
Vivian N. Bui ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Ph Sensor ◽  
Adherens Junction ◽  
Protein Protein Interactions ◽  
Adherens Junction Protein ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Ph Dependent

β-Catenin functions as an adherens junction protein for cell–cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein–protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster. β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R–β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation.

scholarly journals The Role of Posttranslational Modifications in DNA Repair

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Miaomiao Bai ◽  
Dongdong Ti ◽  
Qian Mei ◽  
Jiejie Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Protein Localization ◽  
Complex Structure ◽  
Protein Protein Interactions ◽  
Regulate Protein

The human body is a complex structure of cells, which are exposed to many types of stress. Cells must utilize various mechanisms to protect their DNA from damage caused by metabolic and external sources to maintain genomic integrity and homeostasis and to prevent the development of cancer. DNA damage inevitably occurs regardless of physiological or abnormal conditions. In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, posttranslational modifications (PTMs) can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions. Thus, PTMs in DNA repair should be studied. In this review, we will focus on the current understanding of the phosphorylation, poly(ADP-ribosyl)ation, ubiquitination, SUMOylation, acetylation, and methylation of six typical PTMs and summarize PTMs of the key proteins in DNA repair, providing important insight into the role of PTMs in the maintenance of genome stability and contributing to reveal new and selective therapeutic approaches to target cancers.

scholarly journals The NHR1 Domain of Neuralized Binds Delta and Mediates Delta Trafficking and Notch Signaling

2007 ◽  
Vol 18 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Cosimo Commisso ◽  
Gabrielle L. Boulianne
Keyword(s):  
Plasma Membrane ◽  
Notch Signaling ◽  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Neural Development ◽  
Ring Domain ◽  
Protein Protein Interactions ◽  
Necessary And Sufficient ◽  
Conserved Residue ◽  
Notch Ligands

Notch signaling, which is crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. Here we show that neuralized1, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, we describe a novel isoform of Neuralized and show that it is recruited to the plasma membrane by Delta and that this is mediated by the NHR1 domain. Finally, we show that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, our data demonstrate that NHR domains can function in facilitating protein–protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta.

scholarly journals Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
M Aslam ◽  
H Idrees ◽  
C W Hamm ◽  
Y Ladilov
Keyword(s):  
Endothelial Cells ◽  
Glucose Uptake ◽  
Barrier Function ◽  
Atp Synthesis ◽  
Fold Increase ◽  
Endothelial Barrier ◽  
Synthesis Rate ◽  
Endothelial Barrier Function ◽  
Metabolic Switch ◽  
Barrier Integrity

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

scholarly journals FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB

2019 ◽  
Vol 30 (5) ◽  
pp. 607-621 ◽  
Author(s):  
Manon C. A. Pronk ◽  
Jisca Majolée ◽  
Anke Loregger ◽  
Jan S. M. van Bezu ◽  
Noam Zelcer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Rho Gtpases ◽  
Umbilical Vein ◽  
Cholesterol Biosynthesis ◽  
Endothelial Barrier ◽  
Biosynthesis Pathway ◽  
Endothelial Barrier Function ◽  
Cholesterol Biosynthesis Pathway

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference–based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

scholarly journals MYADM controls endothelial barrier function through ERM-dependent regulation of ICAM-1 expression

2013 ◽  
Vol 24 (4) ◽  
pp. 483-494 ◽  
Author(s):  
Juan F. Aranda ◽  
Natalia Reglero-Real ◽  
Beatriz Marcos-Ramiro ◽  
Ana Ruiz-Sáenz ◽  
Laura Fernández-Martín ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Inflammatory Response ◽  
Barrier Function ◽  
Leukocyte Adhesion ◽  
Cell Junctions ◽  
Membrane Domains ◽  
Filamentous Actin ◽  
Cytokine Tumor Necrosis Factor ◽  
Factor Α

The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. Membrane rafts are ordered assemblies of cholesterol, glycolipids, and proteins that modulate proinflammatory cell signaling and barrier function. In epithelial cells, the MAL family members MAL, MAL2, and myeloid-associated differentiation marker (MYADM) regulate the function and dynamics of ordered membrane domains. We analyzed the expression of these three proteins in human endothelial cells and found that only MYADM is expressed. MYADM was confined in ordered domains at the plasma membrane, where it partially colocalized with filamentous actin and cell–cell junctions. Small interfering RNA (siRNA)-mediated MYADM knockdown increased permeability, ICAM-1 expression, and leukocyte adhesion, all of which are features of an inflammatory response. Barrier function decrease in MYADM-silenced cells was dependent on ICAM-1 expression. Membrane domains and the underlying actin cytoskeleton can regulate each other and are connected by ezrin, radixin, and moesin (ERM) proteins. In endothelial cells, MYADM knockdown induced ERM activation. Triple-ERM knockdown partially inhibited ICAM-1 increase induced by MYADM siRNA. Importantly, ERM knockdown also reduced ICAM-1 expression in response to the proinflammatory cytokine tumor necrosis factor-α. MYADM therefore regulates the connection between the plasma membrane and the cortical cytoskeleton and so can control the endothelial inflammatory response.

P6289Neuregulin-1 improves cardiac function through protecting the loss of endothelial barrier in septic rats via RhoA/ROCK pathway

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
L O N G Wang ◽  
W E N Kang ◽  
Y U E Cheng ◽  
X I Wang
Keyword(s):  
Endothelial Cells ◽  
Survival Rate ◽  
Endothelial Function ◽  
Cardiac Function ◽  
Barrier Function ◽  
Endothelial Barrier ◽  
Intercellular Adhesion Molecule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Endothelial Lining ◽  
Neuregulin 1

Abstract Background The main path of sepsis-induced cardiomyopathy is the loss of endothelial barrier function. Neuregulin-1 (NRG-1) has beneficial effects on endothelial function. Purpose To investigate the effect of NRG-1 treatment on the protection of cardiac endothelial cells and the changes of RhoA/ROCK signaling in sepsis. Methods Rats were randomly divided into three groups: sham, LPS, and NRG-1. After successful induction of sepsis by lipopolysaccharide (LPS, 10mg/kg), rats were administered either a vehicle or recombinant human neuregulin-1 (rhNRG-1, 10μg/kg/d) for one or two days. We recorded their survival rate at 48h after sepsis. Hemodynamic methods were performed to assess cardiac function. We used immunofluorescence assay to detect von Willebrand Factor (vWF). Intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF)levels in serum were measured by enzyme-linked immunosorbent assay (ELISA), and serum nitric oxide (NO) was detected by reductase method. We used transmission electron microscopy to observe changes in myocardial ultrastructure and western blot to assess expression of RhoA and ROCK1 protein. Results Sepsis impaired endothelial function manifested by increased ICAM-1, NO and VEGF levels in serum, NRG-1 treatment could significantly alleviate these increase (P<0.05). Compared with the vehicle, NRG-1 significantly decreased cardiac vascular permeability through increasing the expression of vWF on endothelial lining (P<0.05). Moreover, NRG-1 alleviated damages of ultrastructure of myocardial cells and suppressed the expression of RhoA and ROCK1 protein (P<0.05). Ultimately, NRG-1 improved the survival,and prevented hemodynamic derangement. Levels of endothelial biomarker in serum ICAM-1 (pg/ml) VEGF (pg/ml) NO (pg/ml) Sham 24h 1564.74±94.41 0.84±0.28 203.27±1.81 Sham 48h 322.92±7.92 1.78±0.61 6.57±0.38 LPS 24h 5139.85±284.15† 3.11±0.04† 679.05±78.59† LPS 48h 950.41±25.23†‡ 4.65±0.30† 180.40±30.08†‡ NRG 24h 2772.18±164.45§ 0.30±0.04§ 355.14±23.41§ NRG 48h 578.57±28.97§‡ 1.02±0.45§ 104.67±2.13§‡ †p<0.05 vs. the sham group, §p<0.05 vs. the LPS group and ‡p<0.05 vs. the 24h group. Effect of NRG-1 on endothelial cells Conclusions NRG-1 could alleviate endothelial injury in sepsis by strengthening the barrier function of vascular, reducing the secretion of endothelial-related biomarkers and inhibiting oxidative stress, thus improving cardiac function and survival rate, these effects may base on RhoA/ROCK signaling pathway. These may contribute to reverse the impaired endothelial cells in sepsis in the future. Acknowledgement/Funding The national natural science foundation of China (81772044)

scholarly journals Podocalyxin is required for maintaining blood–brain barrier function during acute inflammation

2019 ◽  
Vol 116 (10) ◽  
pp. 4518-4527 ◽  
Author(s):  
Jessica Cait ◽  
Michael R. Hughes ◽  
Matthew R. Zeglinski ◽  
Allen W. Chan ◽  
Sabrina Osterhof ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Barrier Function ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Focal Adhesions ◽  
Brain Barrier ◽  
Cortical Actin ◽  
Inflammatory Conditions ◽  
Blood Brain

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele ofPodxlin vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood–brain barrier (BBB) inPodxlΔTie2Cremice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primedPodxlΔTie2Cremice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

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