Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity

Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.     

Interaction of plasminogen activator inhibitor-2 and proteasome subunit, beta type 1

The apoptosis protection by plasminogen activator inhibitor-2(PAI-2) is dependent on a 33 amino acid fragment between helix C and D of PAI-2 which is probably due to the interaction of PAI-2 with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 241 amino acids of proteasome (prosome, macropain) subunit, beta type l(PSMβ1) which plays important roles in NF-jjjjjjjjB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interation with PSMβ1.     

Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles

In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.     

Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.     

Heparan sulfate degradation products can associate with oxidized proteins and proteasomes

The S-nitrosylated proteoglycan glypican-1 recycles via endosomes where its heparan sulfate chains are degraded into anhydromannose-containing saccharides by NO-catalyzed deaminative cleavage. Because heparan sulfate chains can be associated with intracellular protein aggregates, glypican-1 autoprocessing may be involved in the clearance of misfolded recycling proteins. Here we have arrested and then reactivated NO-catalyzed cleavage in the absence or presence of proteasome inhibitors and analyzed the products present in endosomes or co-precipitating with proteasomes using metabolic radiolabeling and immunomagnet isolation as well as by confocal immunofluorescence microscopy. Upon reactivation of deaminative cleavage in T24 carcinoma cells, [(35)S]sulfate-labeled degradation products appeared in Rab7-positive vesicles and co-precipitated with a 20 S proteasome subunit. Simultaneous inhibition of proteasome activity resulted in a sustained accumulation of degradation products. We also demonstrated that the anhydromannose-containing heparan sulfate degradation products are detected by a hydrazide-based method that also identifies oxidized, i.e. carbonylated, proteins that are normally degraded in proteasomes. Upon inhibition of proteasome activity, pronounced colocalization between carbonyl-staining, anhydro-mannose-containing degradation products, and proteasomes was observed in both T24 carcinoma and N2a neuroblastoma cells. The deaminatively generated products that co-precipitated with the proteasomal subunit contained heparan sulfate but were larger than heparan sulfate oligosaccharides and resistant to both acid and alkali. However, proteolytic degradation released heparan sulfate oligosaccharides. In Niemann-Pick C-1 fibroblasts, where deaminative degradation of heparan sulfate is defective, carbonylated proteins were abundant. Moreover, when glypican-1 expression was silenced in normal fibroblasts, the level of carbonylated proteins increased raising the possibility that deaminative heparan sulfate degradation is involved in the clearance of misfolded proteins.     

Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells.

Recent studies have proposed that post-translational degradation of apolipoprotein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)-associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that recognize both alpha- and beta-subunits of the proteasome co-localized in the ER with specific domains of apoB. The mechanism of apoB degradation in the ER by the ubiquitin-proteasome pathway was studied using pulse-chase labeling and digitonin-permeabilized cells. ApoB in permeabilized cells incubated at 37 degrees C in buffer alone was relatively stable. When permeabilized cells were incubated with both exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of ubiquitin-proteasome factors, >50% of [3H]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabilized cells was much more rapid than that of extracted [3H]apoB incubated with RRL and ATP in vitro. The degradation of apoB was reduced by clasto-lactacystin beta-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesized apoB in the ER is accessible to the cytoplasmic ubiquitin-proteasome pathway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.     

Evidence for serpinB2-independent protection from TNF-alpha-induced apoptosis

Clade B serine proteinase inhibitors (serpins) are intracellular proteins, whereas most of their identified targets are extracellular. A proposed intracellular role for these inhibitors is protection from apoptosis. We investigated the contribution of serpinB2 (plasminogen activator inhibitor-2, PAI-2) activity in TNF-alpha-induced apoptosis. PAI-2 is expressed in many normal and transformed cell types, particularly after stimulation with inflammatory cytokines. PAI-2 has been linked to protection from TNF-alpha-induced apoptosis, and a stabilizing interaction with the retinoblastoma protein (Rb1) has been proposed. We examined the activity of PAI-2 in TNF-alpha-induced apoptosis using HeLa, Isreco-1 and HT1080 cell lines. Stimulation with TNF-alpha protected each cell type from apoptosis induced by TNF-alpha and cycloheximide. Protection correlated with an increase in PAI-2 expression in IS-1 and HT1080 cells but not in HeLa cells where PAI-2 mRNA and protein were undetectable. PAI-2 was overexpressed in each cell type but gave no protection from TNF-alpha-induced apoptosis measured by cell viability, annexinV binding and caspase-3/7 activity. We detected wild-type Rb1, unchanged TNF receptor levels and induction of other apoptosis-protective factors in all cell types. In conclusion, elevated PAI-2 levels do not protect cells from TNF-alpha-induced apoptosis, and the protective effect of prior stimulation with TNF-alpha does not require PAI-2.     

Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential

Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.     

Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.     

Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells

Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.     

Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin.

Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators.     

Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin

The action of oxidatively modified low-density lipoprotein (oxLDL) on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. OxLDL was shown here to elicit oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2',7'-dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. A 170-kDa polypeptide carbonylated upon exposure of cells to oxLDL was identified by immunoprecipitation as EGF receptor. Immunocytochemical visualization by confocal microscopy revealed the highest levels of modified proteins in the perinuclear region. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of *NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be both carbonylated and nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor L-NIO. These oxLDL-elicited changes in vascular endothelial cells described were suppressed by (-)-epicatechin, a dietary polyphenol, which inhibited NADPH oxidase activity in these cells.     

Modulation of the chymotrypsin-like activity of the 20S proteasome by intracellular redox status: effects of glutathione peroxidase-1 overexpression and antioxidant drugs

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.     

Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics

We have developed a heterobifunctional all-small molecule PROTAC (PROteolysis TArgeting Chimera) capable of inducing proteasomal degradation of the androgen receptor. This cell permeable PROTAC consists of a non-steroidal androgen receptor ligand (SARM) and the MDM2 ligand known as nutlin, connected by a PEG-based linker. The SARM-nutlin PROTAC recruits the androgen receptor to MDM2, which functions as an E3 ubiquitin ligase. This leads to the ubiquitination of the androgen receptor, and its subsequent degradation by the proteasome. Upon treatment of HeLa cells with 10microM PROTAC for 7h, we were able to observe a decrease in androgen receptor levels. This degradation is proteasome dependent, as it is mitigated in cells pre-treated with 10microM epoxomicin, a specific proteasome inhibitor. These results have implications for the potential study and treatment of various cancers with increased androgen receptor levels.     

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.     

COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.