Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

m6A Methyltransferase, WTAP, Promotes Cancer Progression in Laryngeal Squamous Cell Carcinoma by Regulating PLAU Stability.

OBJECTIVE: Laryngeal squamous cell carcinoma (LSCC) is a malignancy originating from laryngeal squamous cell lesions. Wilm's tumor 1-associated protein (WTAP)-mediated N6-methyladenosine (m6A) modification has been verified to stimulate the progression of numerous cancers, except for LSCC. This study was aimed at exploring the role of WTAP and its mechanism of action in LSCC. METHODS: The expression of WTAP and plasminogen activator urokinase (PLAU) mRNAs in LSCC tissues and cells was quantified using qRT-PCR. Western blotting was performed to estimate PLAU levels in LSCC cells. The relationship between WTAP and PLAU was ascertained using luciferase reporter and methylated-RNA immunoprecipitation (Me-RIP) assays. Functionally, the interaction of WTAP with PLAU in LSCC cells was investigated using CCK-8, EdU, and Transwell assays. RESULTS: The expression of WTAP and PLAU was increased in LSCC, and was positively correlated. WTAP regulated PLAU stability in an m6A-dependent manner. WTAP deficiency suppressed the migration, invasion, and proliferation of LSCC cells. Overexpression of PLAU rescued the phenotype induced by WTAP knockdown in vitro. CONCLUSIONS: These results indicate that WTAP mediates the m6A modification of PLAU to accelerate the growth, migration, and invasion of cells in LSCC. To our knowledge, this is the first report to clarify the functions of WTAP in LSCC and the underlying mechanisms in detail. Based on these findings, we suggest that WTAP may serve as a therapeutic target for LSCC.

PLAU contributes to the development of cholangiocarcinoma via activating NF-κB signaling pathway.

Cholangiocarcinoma (CCA) is a type of epithelial cancer with poor outcomes and late diagnosis. Accumulating evidence has demonstrated the promoting role of plasminogen activator, urokinase (PLAU) in several tumor types, while its function in CCA is largely unknown. The expression of PLAU in CCA was determined by data from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database and further confirmed in human tissues using immunohistochemical (IHC) staining. Moreover, PLAU-silencing CCA cell models were constructed for subsequent functional assays in vitro and in vivo. PLAU expression in CCA was significantly higher than that in normal tissues. High PLAU expression was positively correlated with poor patients' survival. PLAU knockdown remarkably suppressed proliferation and migration of CCA cells, whereas enhanced apoptosis. Consistently, tumor growth in mice injected with PLAU-silencing CCA cells was also impaired. Furthermore, we revealed that the activation of NF-κB signaling was required for PLAU-induced malignant phenotypes of CCA cells. Inhibiting the high expression of PLAU in CCA may be a potential entry point for targeted therapy in CCA patient.

Extracellular vesicle-derived LINC00511 promotes glycolysis and mitochondrial oxidative phosphorylation of pancreatic cancer through macrophage polarization by microRNA-193a-3p-dependent regulation of plasminogen activator urokinase.

OBJECTIVE: The involvement of tumor-derived extracellular vesicles (EVs) in macrophage polarization has been reported. In our present study, we tried to discuss the regulatory role of LINC00511 encapsulated in pancreatic cancer (PCa) cell-derived EVs in the development and progression of PCa. METHODS: EVs from PCa cell line BxPC-3 culture medium were collected and subsequently identified by electron microscopy and nanoparticle tracking analysis. The expression pattern of LINC00511 in PCa cell-derived EVs was determined. The interaction among LINC00511, microRNA-193a-3p, and plasminogen activator urokinase (PLAU) was explored. After co-culture of PCa cell-derived EVs with macrophages, the regulatory roles of LINC00511 in macrophage polarization, PCa cell functions, glucose consumption, lactate production, glycolysis, and mitochondrial oxidative phosphorylation were investigated. RESULTS: PCa cell line BxPC-3 had highly expressed LINC00511 and LINC00511 could be internalized by macrophages. LINC00511 affected macrophage polarization through miR-193a-3p-dependent regulation of PLAU expression. Besides, EV-derived LINC00511 accelerated glycolysis and promoted mitochondrial oxidative phosphorylation of PCa cells through macrophage polarization, thus inducing invasion and migration of PCa cells. CONCLUSION: LINC00511 encapsulated in PCa cell-derived EVs facilitates glycolysis of PCa cells through regulation of macrophage polarization in the tumor microenvironment.

Proteomics analysis uncovers plasminogen activator PLAU as a target of the STING pathway for suppression of cancer cell migration and invasion.

The stimulator of interferon genes (STING) pathway is vital for immune defense against pathogen invasion and cancer. Although ample evidence substantiates that the STING signaling pathway plays an essential role in various cancers via cytokines, no comprehensive investigation of secretory proteins regulated by the STING pathway has been conducted hitherto. Herein, we identify 24 secretory proteins significantly regulated by the STING signaling pathway through quantitative proteomics. Mechanistic analyses reveal that STING activation inhibits the translation of urokinase-type plasminogen activator (PLAU) via the STING-PERK-eIF2α signaling axis. PLAU is highly expressed in a variety of cancers and promotes the migration and invasion of cancer cells. Notably, the activation of STING inhibits cancer cell migration and invasion by suppressing PLAU. Collectively, these results provide novel insights into the anticancer mechanism of the STING pathway, offering a theoretical basis for precision therapy for this patient population.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses.

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell-bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands.

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19 ± 0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

Astrocyte tissue plasminogen activator expression during brain development and its role in pyramidal neuron neurite outgrowth.

The serine protease tissue plasminogen activator (tPA), encoded by the gene Plat, exerts a wide range of proteolysis-dependent and proteolysis-independent functions. In the developing brain, tPA is involved in neuronal development via the modulation of the proteolytic degradation of the extracellular matrix (ECM). Both lack of and excessive tPA are associated with neurodevelopmental disorders and with brain pathology. Astrocytes play a major role in neurite outgrowth of developing neurons as they are major producers of ECM proteins and ECM proteases. In this study we investigated the expression of Plat in developing and mature hippocampal and cortical astrocytes of Aldh1l1-EGFP-Rpl10a mice in vivo following Translating Ribosome Affinity Purification (TRAP) and the role of tPA in modulating astrocyte-mediated neurite outgrowth in an in vitro astrocyte-neuron co-culture system. We show that Plat is highly enriched in astrocytes in the developing, but not in the mature, hippocampus and cortex. Both the silencing of tPA expression in astrocytes and astrocyte exposure to recombinant tPA reduce neuritogenesis in co-cultured hippocampal neurons. These results suggest that astrocyte tPA is involved in modulating neuronal development and that tight control of astrocyte tPA expression is important for normal neuronal development, with both experimentally elevated and reduced levels of this proteolytic enzyme impairing neurite outgrowth. These results are consistent with the hypothesis that the ECM, by serving as adhesive substrate, enables neurite outgrowth, but that controlled proteolysis of the ECM is needed for growth cone advancement.

Pivotal Role of Corneal Fibroblasts in Progression to Corneal Ulcer in Bacterial Keratitis.

The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment. Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.

Precision targeting of the plasminogen activator inhibitor-1 mechanism increases efficacy of fibrinolytic therapy in empyema.

Plasminogen activator inhibitor-1 (PAI-1) is an endogenous irreversible inhibitor of tissue-type (tPA) and urokinase (uPA) plasminogen activators. PAI-1-targeted fibrinolytic therapy (PAI-1-TFT) is designed to decrease the therapeutic dose of tPA and uPA, attenuating the risk of bleeding and other complications. Docking site peptide (DSP) mimics the part of the PAI-1 reactive center loop that interacts with plasminogen activators, thereby affecting the PAI-1 mechanism. We used DSP for PAI-1-TFT in two rabbit models: chemically induced pleural injury and Streptococcus pneumoniae induced empyema. These models feature different levels of inflammation and PAI-1 expression. PAI-1-TFT with DSP (2.0 mg/kg) converted ineffective doses of single chain (sc) tPA (72.5 µg/kg) and scuPA (62.5 µg/kg) into effective ones in chemically induced pleural injury. DSP (2.0 mg/kg) was ineffective in S. pneumoniae empyema, where the level of PAI-1 is an order of magnitude higher. DSP dose escalation to 8.0 mg/kg resulted in effective PAI-1-TFT with 0.25 mg/kg sctPA (1/8th of the effective dose of sctPA alone) in empyema. There was no increase in the efficacy of scuPA. PAI-1-TFT with DSP increases the efficacy of fibrinolytic therapy up to 8-fold in chemically induced (sctPA and scuPA) and infectious (sctPA) pleural injury in rabbits. PAI-1 is a valid molecular target in our model of S. pneumoniae empyema in rabbits, which closely recapitulates key characteristics of empyema in humans. Low-dose PAI-1-TFT is a novel interventional strategy that offers the potential to improve fibrinolytic therapy for empyema in clinical practice.

Plasminogen Activators in Neurovascular and Neurodegenerative Disorders.

The neurovascular unit (NVU) is a dynamic structure assembled by endothelial cells surrounded by a basement membrane, pericytes, astrocytes, microglia and neurons. A carefully coordinated interplay between these cellular and non-cellular components is required to maintain normal neuronal function, and in line with these observations, a growing body of evidence has linked NVU dysfunction to neurodegeneration. Plasminogen activators catalyze the conversion of the zymogen plasminogen into the two-chain protease plasmin, which in turn triggers a plethora of physiological events including wound healing, angiogenesis, cell migration and inflammation. The last four decades of research have revealed that the two mammalian plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), are pivotal regulators of NVU function during physiological and pathological conditions. Here, we will review the most relevant data on their expression and function in the NVU and their role in neurovascular and neurodegenerative disorders.

Fibrinolysis: A Primordial System Linked to the Immune Response.

The fibrinolytic system provides an essential means to remove fibrin deposits and blood clots. The actual protease responsible for this is plasmin, formed from its precursor, plasminogen. Fibrin is heralded as it most renowned substrate but for many years plasmin has been known to cleave many other substrates, and to also activate other proteolytic systems. Recent clinical studies have shown that the promotion of plasmin can lead to an immunosuppressed phenotype, in part via its ability to modulate cytokine expression. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allows plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, a multitude of pathogens can also express their own plasminogen activators, or contain surface proteins that provide binding sites host plasminogen. Plasmin formed under these circumstances also empowers these pathogens to modulate host immune defense mechanisms. Phylogenetic studies have revealed that the plasminogen activating system predates the appearance of fibrin, indicating that plasmin did not evolve as a fibrinolytic protease but perhaps has its roots as an immune modifying protease. While its fibrin removing capacity became apparent in lower vertebrates these primitive under-appreciated immune modifying functions still remain and are now becoming more recognised.

Modulation of the inflammatory environment by spermatozoa through regulation of transforming growth factor beta in porcine uterine epithelial cells.

This study investigated the changes in the mRNA expression of transforming growth factor beta (TGF-ß), plasminogen activators (PAs), and interleukin (IL) caused by sperm, as well as the regulatory mechanism of PA activity through TGF-ß, in porcine uterine epithelial cells. The cells were isolated from the uterine horn of pig and co-incubated with Percoll-separated boar sperm (45% or 90%), or TGF-ß for 24 h. The mRNA expression of TGF-ß isoforms (TGF-ß1, 2 and 3) and their receptors (TGF-ß R1 and R2), PAs (urokinase-type, uPA; tissue-type, tPA; uPA receptor, uPAR; type 1 PA inhibitor, PAI-1), IL-6 and IL-8 was analyzed using real-time PCR. Supernatant was used to measure PA activity. Co-incubation with sperm from the 90% Percoll layer increased TGF-ß1 mRNA, whereas TGF-ß2 and TGF-ß3 were decreased (P < 0.05). However, both TGF-ßRs were not changed by the presence of the spermatozoa. Expression of tPA, PAI-1, IL-6, and IL-8 mRNA was down-regulated by 90% Percoll-separated sperm (P < 0.05), and sperm from 45% Percoll increased uPA expression (P < 0.05). TGF-ß decreased tPA and IL-8 mRNA expression, and increased uPAR and PAI-1 mRNA (P < 0.05). The suppressive effect of TGF-ß on PA activity was blocked by Smad2/3 and JNK1/2 signaling inhibitors (P < 0.05). In conclusion, sperm separated in 90% in porcine uterus could suppressed inflammation via modulation of TGF-ß and down-regulation of PAs and ILs. Therefore, the regulatory mechanism of inflammation by sperm in the porcine uterus could be associated with interactions between numerous cytokines including TGF-ß.

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Genome sequencing, purification, and biochemical characterization of a strongly fibrinolytic enzyme from Bacillus amyloliquefaciens Jxnuwx-1 isolated from Chinese traditional douchi.

A strongly fibrinolytic enzyme was purified from Bacillus amyloliquefaciens Jxnuwx-1, found in Chinese traditional fermented black soya bean (douchi). The molecular mass of the enzyme, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. The optimal pH and temperature for the enzyme were 7.6 and 41°C, respectively. The enzyme was inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, Fe3+, and Fe2+. The highest affinity exhibited by the enzyme was towards N-Succinyl-Ala-Ala-Pro-Phe-pNA. These results indicated that it is a subtilisin-like serine metalloprotease. The enzyme degraded both fibrinogen and fibrin, displaying its highest degrading activity towards the Aα-chains followed by Bß chains and Cγ chains. The enzyme was also activated by plasminogen, indicating its ability to degrade fibrinogen and fibrin in two ways: (a) by activating plasminogen conversion into plasmin, or (b) by direct hydrolysis. It degraded thrombin, suggesting that it may act as an anticoagulant to prevent thrombosis. Taken together, our results indicate the potential of this enzyme in controlling cardiovascular disease.

Moderate Intensity Static Magnetic Fields Prevent Thrombus Formation in Rats and Mice.

We established three types of thrombosis models to explore the effects of the static magnetic field (SMF) on thrombosis in rats and mice with three different MF intensities. In the carrageenan-induced thrombosis model in rats, the SMF treatments reduced the black tail length of rats, extracorporeal thrombus, and the mass of wet and dry thrombus, and improved the coagulation index value. In FeCl3 -induced arterial thrombosis model in rats, the SMF treatment showed some anti-thrombotic effects. More specifically, the SMF treatment affected rodent blood pressure, plasma plasminogen activator inhibitor, tissue-type plasminogen activator, thrombus mass, and thrombus protein content. In the adrenaline-induced thrombosis model in mice, the SMF treatment had certain effects on the diameter and blood flow velocity of mouse auricle microcirculation in fine veins and arteries. Overall, the highest MF intensities we tested, 20-150 mT, showed a trend of anti-thrombotic effect, indicating that the moderate-intensity SMF might serve as a potential treatment for clot-related diseases in the future. Bioelectromagnetics. 2020;41:52-62 © 2019 Bioelectromagnetics Society.

Changes in uPA, PAI-1, and TGF-ß Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC).

The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-ß1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-ß1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-ß1 synthesis and that TGF-ß1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-ß during the co-culture of breast cancer cells with MSC.

[Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf].

OBJECTIVE: To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. METHODS: Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). RESULTS: Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 µg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. CONCLUSIONS: Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

The ß-domain of streptokinase affects several functionalities, including specific/proteolytic activity kinetics.

Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G Streptococcus (SKA/SKC/SKG) is composed of three domains: SKα, SKß and SKγ. Previous domain-swapping studies between SK1/SK2b-cluster variants revealed that SKß plays a major role in the activation of human Pg. Here, we carried out domain-swapping between skcg-SK/SK2-cluster variants to determine the involvement of SKß in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen-bound Pg activation and α2 -antiplasmin resistance. Our results indicate that SKß has a minor to determining role in these diverse functionalities for skcg-SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKß and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin-specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.