scholarly journals Integrin β3, a RACK1 interacting protein, is required for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells

2020 ◽  
Author(s):  
Chao Yang ◽  
Rui Lan ◽  
Xiaochun Wang ◽  
Qian Zhao ◽  
Xidan Li ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Target Genes ◽  
Activated Protein C ◽  
Respiratory Syndrome Virus ◽  
Interacting Protein ◽  
Pig Production ◽  
Interaction Mechanisms ◽  
Host Pathogen Interaction ◽  
Integrin Β3

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-κB activation in Marc-145 cells. Here we identified a membrane protein, integrin β3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-κB activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-κB activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID50 titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-κB target genes during PRRSV infection, and that they regulate each other. Our data indicate that ITGB3, presumably as a co-receptor, plays an imperative role for PRRSV infection and NF-κB activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-κB and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host–pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the pathogens in pig production worldwide. Several cell surface receptors, such as heparan sulphate, sialoadhesin, vimentin and CD163, were identified to be involved in PRRSV infection in porcine alveolar macrophages (PAMs). We identified a cell surface protein, integrin β3 (ITGB3), as an interacting protein with receptor of activated protein C kinase 1 (RACK1) from Marc-145 cells. ITGB3 interacts with RACK1 and facilitates PRRSV infection and NF-κB activation in Marc-145 cells, presumably as a co-receptor of CD136 or vimentin. Both ITGB3 and RACK1 were NF-κB target genes, and they regulate each other. The activation of NF-κB and the transcription of its downstream genes are beneficial for PRRSV infection/replication. The novel findings would advance our elaborated understanding of the molecular host–pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3-RACK1-NF-κB axis as a potential therapeutic target for PRRS control.

scholarly journals Proteomic Analysis of Leishmania donovani Membrane Components Reveals the Role of Activated Protein C Kinase in Host-Parasite Interaction

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1194
Author(s):  
Sandeep Verma ◽  
Deepak Kumar Deep ◽  
Poonam Gautam ◽  
Ruchi Singh ◽  
Poonam Salotra
Keyword(s):  
Membrane Proteins ◽  
Protein C ◽  
Leishmania Donovani ◽  
Parasitic Infection ◽  
Activated Protein C ◽  
Host Cells ◽  
Filamin A ◽  
Related Factors ◽  
Interacting Protein ◽  
Host Parasite

Visceral leishmaniasis (VL), mainly caused by the Leishmania donovani parasitic infection, constitutes a potentially fatal disease, for which treatment is primarily dependent on chemotherapy. The emergence of a resistant parasite towards current antileishmanial agents and increasing reports of relapses are the major concerns. Detailed research on the molecular interaction at the host-parasite interface may provide the identification of the parasite and the host-related factors operating during disease development. Genomic and proteomic studies highlighted several essential secretory and cytosolic proteins that play vital roles during Leishmania pathogenesis. The aim of this study was to identify membrane proteins from the Leishmania donovani parasite and the host macrophage that interact with each other using 2-DE/MALDI-TOF/MS. We identified membrane proteins including activated protein C kinase, peroxidoxin, small myristoylated protein 1 (SMP-1), and cytochrome C oxidase from the parasite, while identifying filamin A interacting protein 1(FILIP1) and β-actin from macrophages. We further investigated parasite replication and persistence within macrophages following the macrophage-amastigote model in the presence or absence of withaferin (WA), an inhibitor of activated C kinase. WA significantly reduced Leishmania donovani replication within host macrophages. This study sheds light on the important interacting proteins for parasite proliferation and virulence, and the establishment of infection within host cells, which can be targeted further to develop a strategy for chemotherapeutic intervention.

A Novel Mechanism of Endothelial Protein C Receptor Release, in Microparticulate Form, by Activated Protein C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1923-1923
Author(s):  
Margarita Perez-Casal ◽  
Kenji Fukudome ◽  
Cheng Hock Toh
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Umbilical Vein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Endothelial Protein C Receptor ◽  
Factor Va ◽  
Metalloproteinase Inhibitors ◽  
Cytokine Stimulation ◽  
Umbilical Vein Endothelial Cells

Abstract Activated protein C (APC) administration is now used for treating patients with severe sepsis. We investigated its effect on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release from the cell surface. Exposure of human umbilical vein endothelial cells or monocytes to APC (from physiological levels of 0.5 up to 100nM) resulted in the increasing release of EPCR-containing microparticles (EPCR-MP), as demonstrated by confocal microscopy. Further characterisation through flow cytometry showed a concomitant fall in EPCR levels from the cell surface. This release of EPCR could not be inhibited by the metalloproteinase inhibitors 1, 10-phenanthroline or Ro31-9790, unlike soluble EPCR (sEPCR) that is metalloproteinase cleaved at the cell surface following thrombin or pro- inflammatory cytokine stimulation. Western blotting confirmed the molecular weight of EPCR-MP to be identical to the full-length membrane form (49 kD) and different from sEPCR (45 kDa). APC was also bound to EPCR-MP and could be quantified by ELISA using EPCR capture and APC detection by chromogenic substrate, S2366. Using an initial factor Va incubation step followed by a prothrombinase assay, the APC bound to EPCR-MP could significantly reduce thrombin generation. This was abrogated in the presence of excess α1-antitrypsin, an APC inhibitor. By contrast, APC bound to sEPCR could no longer inactivate factor Va. Further characterisation showed the APC induction of EPCR-MP to be time dependent with increasing release over 24 hours, as quantified by ELISA. The phenomenon also required the active site of APC. Neither protein C, heat-inactivated or D-Phe-Pro-Arg-chloromethylketone-blocked APC could induce EPCR-MP formation. Co-incubation with hirudin (6mM) did not alter the APC effect and excluded any role of contaminating thrombin. This novel observation provides new insights into the consequences of APC therapy in the septic patient as well as demonstrating for the first time that there can be 2 circulating forms of EPCR. Unlike sEPCR however, EPCR-MP can facilitate and potentially disseminate the anticoagulant activity of bound APC.

scholarly journals Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function

Blood ◽  
2017 ◽  
Vol 130 (12) ◽  
pp. 1445-1455 ◽  
Author(s):  
Thati Madhusudhan ◽  
Hongjie Wang ◽  
Sanchita Ghosh ◽  
Wei Dong ◽  
Varun Kumar ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Protein C ◽  
Target Genes ◽  
Activated Protein C ◽  
Regulatory Subunits ◽  
Downstream Signaling ◽  
Unfolded Protein ◽  
Filtration Barrier ◽  
Transcriptional Regulatory ◽  
Protein Response

Abstract Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85β. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin signaling intermediates may be a feasible therapeutic approach ameliorating defective insulin signaling.

scholarly journals Endothelial Protein C Receptor (EPCR), Protease Activated Receptor-1 (PAR-1) and Their Interplay in Cancer Growth and Metastatic Dissemination

Cancers ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 51 ◽  
Author(s):  
Marek Z. Wojtukiewicz ◽  
Dominika Hempel ◽  
Ewa Sierko ◽  
Stephanie C. Tucker ◽  
Kenneth V. Honn
Keyword(s):  
Cell Surface ◽  
Cancer Patients ◽  
Cancer Progression ◽  
Protein C ◽  
Critical Role ◽  
Activated Protein C ◽  
Cancer Growth ◽  
Endothelial Protein C Receptor ◽  
Surface Receptors ◽  
Protease Activated Receptor 1

Endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR-1) by themselves play important role in cancer growth and dissemination. Moreover, interactions between the two receptors are essential for tumor progression. EPCR is a cell surface transmembrane glycoprotein localized predominantly on endothelial cells (ECs). It is a vital component of the activated protein C (APC)—mediated anticoagulant and cytoprotective signaling cascade. PAR-1, which belongs to a family of G protein–coupled cell surface receptors, is also widely distributed on endothelial and blood cells, where it plays a critical role in hemostasis. Both EPCR and PAR-1, generally considered coagulation-related receptors, are implicated in carcinogenesis and dissemination of diverse tumor types, and their expression correlates with clinical outcome of cancer patients. Existing data explain some mechanisms by which EPCR/PAR-1 affects cancer growth and metastasis; however, the exact molecular basis of cancer invasion associated with the signaling is still obscure. Here, we discuss the role of EPCR and PAR-1 reciprocal interactions in cancer progression as well as potential therapeutic options targeted specifically to interact with EPCR/PAR-1-induced signaling in cancer patients.

Activated protein C generation is greatly decreased in plasma from newborns compared to adults in the presence or absence of endothelium

2004 ◽  
Vol 91 (02) ◽  
pp. 238-247 ◽  
Author(s):  
Sanjay Patel ◽  
Christoph Male ◽  
Leslie Berry ◽  
Lesley Mitchell ◽  
Anthony Chan
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Umbilical Cord ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Age Related ◽  
Adult Plasma ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryActivated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation ± endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor ± thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37oC. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-α1antitrypsin (APC-α1AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APCPCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-α2macroglobulin (APC-α2M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-α1AT were decreased in newborn compared to adult plasma on plastic. However, APC-α2M made up a larger fraction of inhibitor complexes in newborn plasma. On endothelium, significantly more APC, APC-PCI and APC-α1AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-α1AT were also reduced and total APC-α2M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-α1AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.

SIMULTANEOUS PRODUCTION OF ENDOTHELIAL CELL-DERIVED PROTEIN S AND FACTOR V, AND INACTIVATION OF FACTOR Va AT THE ENDOTHELIAL CELL SURFACE

1987 ◽  
Author(s):  
P v d Waart ◽  
K T Preissner ◽  
U Delvos ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Conditioned Medium ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Endothelial Cell Surface ◽  
Factor Va

Several proteins synthesized and expressed by endothelial cells are involved in the regulation of coagulation. The synthesis and expression of factor V and protein S has been demonstrated in independent studies. The present work evaluates the simultaneous synthesis and expression of bovine factor V and protein S and the effect of endothelial protein S on the inactivation of endothelial factor Va by activated protein C. The accumulation of both proteins in conditioned medium was detected by SDS-PAGE followed by immunoblotting, and their activities were tested by functional assays. The synthesis of protein S and factor V per 105 cells over 24 h amounted up to 2 ng protein S and 440 ng factor V, respectively. The addition of thrombin did not increase the yield of synthesized cofactors. Thrombin did neither proteolyse protein S on endothelial cells nor in a purified system in the presence of thrombomodulin and calcium ions. Factor V was secreted partly in its activated form as evidenced by the appearance of active intermediates with M = 220,000-280,000 on immunoblots as well as by only a three-Fold further activation of factor V/Va following addition of thrombin. The rate constant for the inactivation of factor Va by activated protein C was only two-fold higher for factor Va derived from cells cultured in the presence of vitamin K as compared in the presence of warfarin. For the inactivation of comparable factor Va concentrations in conditioned medium a 10-fold higher and on endothelial cells a 40-fold higher concentration of activated protein C was required to obtain similar inactivation rates of factor Va as compared to a purified system. These results suggest that resting endothelial cells contain a factor V activator, and that a regulatory mechanism is operative on the endothelial cell surface that suppresses the inactivation potential of activated protein C/ protein S.

MODULATION OF ENDOTHELIAL CELL COAGULANT PROPERTIES

1987 ◽  
Author(s):  
David M Stern ◽  
Sara Rimon ◽  
Todd Scott ◽  
Peter P Nawroth
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Protein C ◽  
Interleukin 1 ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Ix ◽  
Receptor Expression ◽  
Factor X ◽  
Factor Ixa

As the cells forming the luminal vascular surface, endothelium is strategically located to play a role in the regulation of coagulation. Participation of endothelium in coagulation involves specific receptors on the cell surface functioning at the level of initiation and propagation of hemostatic reactions. In the anticoagulant protein C pathway, for example, the receptor thrombomodulin initiates thrombin-mediated activation of protein C and a binding site for protein S on bovine endothelium promotes assembly of the functional activated protein C/protein S complex. Endothelium also synthesizes, stores and releases functional protein S constitutively and in response to specific stimuli such as norepinephrine.Since activation of protein C requires thrombin formation in proximity to the vessel wall, we have examined procoagulant reactions on the endothelial cell surface. Endothelium provides a receptor for Factor IX/IXa which is relatively selective for the enzyme form and facilitates Factor IXa-VIII-mediated activation of Factor X. Half-maximal Factor Xa formation occurs at a Factor IXa concentration of 0.4nM on endothelium, whereas lOnM is required on liposomes. This concentration of Factor IXa corresponds to that which results in half-maximal occupancy of endothelial cell Factor IXa binding sites in the presence of Factors VIII and X, thus correlating kinetics and binding measurements. Crosslinking and ligand blotting studies have shown that the receptor is a protein with a molecular weight of ∼160,000. The clinical significance of this receptor is suggested by the moderately severe bleeding disorder observed in a patient with hemophilia B due to an abnormal Factor IX molecule, Factor IXalabama (Factor IXala). Although the coagulant activity of Factor IXala is only mildly decreased on phospholipids, it is severely impaired on endothelium. The affinity of Factor IXala for the endothelial cell Factor X activation complex is decreased by 20-fold compared with the normal enzyme and the binding affinity is similarly decreased. Since the molecular defect in Factor IXala has been previously shown to consist of a single point mutation in the growth factor domain, this indicates a role for the growth factor domain in receptor, recognition.The picture of endothelial cell coagulant properties which emerges from these and other studies is one in which endothelium has either an anticoagulant or procoagulant potential, depending on modulation of receptor expression and release of secreted products. In the quiescent state, anticoagulant mechanisms predominate with only limited amounts of procoagulant activity: there is little tissue factor activity and only a basal level of receptors for Factor IX/lXa. Activation of endothelium by Tumor Necrosis Factor (TNF) or Interleukin 1 can shift this balance. Tissue factor synthesis and expression occurs in a dose-dependent manner, being half-maximal at a TNF concentration of about 150pM. TNF also increases the number of Factor IX/lXa binding sites. Concomitant with enhancement of endothelial cell procoagulant properties is a suppression of cell surface cofactor activity for the anticoagulant protein C pathway. Endothelial cell-dependent, thrombin-mediated activated protein C formation is decreased by 70-80% and activated protein C-protein S-mediated Factor Va inactivation decreases by over 90%. Following the in vivo infusion of Interleukin 1, similar changes in endothelial cell coagulant properties were observed on aortic segments with fibrin deposition occurring on the functionally altered, but morphologically intact endothelium. This modulation of endothelial cell coagulant properties could underlie the prothrombotic state associated with inflammatory disorders and could also explain the recently observed selective intravascular thrombosis of tumor vasculature seen in vivo in meth A sarcomas after administration of TNF.Thus, although endothelium was initially felt to be hemostatically inert, this apparent lack of activity actually masks a delicate balance of procoagulant and anticoagulant mechanisms. The balance can be effectively shifted by physiologic mediators, such as monokines, which alter receptor expression on the endothelial cell surface. Changes in endothelial cell hemostatic properties may be an early indicator of vessel wall disease and underlie the pathogenesis of localized thrombotic processes.

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