Mechanism Of Action And Regulation Of Membrane Serine Protease Prostasin In The Prostate And Prostate Cancer

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation of the epidermal growth factor receptor (EGFR). As a result, the cellular response to EGF was reduced as shown by the down-regulation of EGF-stimulated Erk1/2 phosphorylation. The expression of Slug, urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and granulocyte-macrophage colony stimulating factor (GM-CSF) was also down-regulated by prostasin re-expression in the PC-3 cells. Co-expression of prostasin and its activating protease matriptase with EGFR in FT-293 cells induces an apparent proteolytic cleavage of the EGFR in the extracellular domain at two specific sites, generating two N-terminally truncated EGFR fragments, named EGFR135 and EGFR110. The EGFR110 is constitutively tyrosine-phosphorylated, and in its presence the phosphorylation of downstream signaling molecules including Erk1/2 and Akt is increased under serum-free conditions. Neither EGFR135 nor EGFR110 is responsive to EGF stimulation. Deletions of the EGFR extracellular domain (ECD) were generated to map the matriptase-prostasin cleavage sites. Two candidate sites were localized to regions AA1-273 and AA273-410. These data support a mechanism of action for the matriptase-prostasin epithelial extracellular serine protease activation cascade by proteolytically modulating the EGF-EGFR signaling. Prostasin gene expression is down-regulated in high-grade and hormone-refractory prostate cancers. We investigated the mechanisms by which androgens regulate prostasin expression in the prostate and prostate cancer. We treated the LNCaP human prostate cancer cells with dihydrotestosterone (DHT) and measured the mRNA expression of prostasin and potential transcription regulators of prostasin predicted by interrogation of the prostasin gene promoter sequence. Prostasin mRNA expression in the LNCaP cells was not responsive to DHT treatment. DHT marginally up-regulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of a prostasin promoter-reporter and SREBP cDNA in HEK-293 cells resulted in stimulation of the promoter activity at ~2 fold by SREBP-1c, and up to 6 fold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of the promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2 s stimulation of the prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 cells resulted in up-regulation of the endogenously expressed prostasin while transfection of a SLUG cDNA in the LNCaP cells repressed prostasin expression. Multiple SREBP-2 binding sites, known as sterol regulatory elements (SRE s), were identified at positions -897, -538, +8, +71, and +98 (named SRE-897, SRE-538, SRE+8, SRE+71, and SRE+98) in the human prostasin gene promoter. Mutagenesis of the five SRE s was carried out to evaluate their roles in SREBP-2 up-regulation of prostasin. SRE+98, a novel functional sterol regulatory element was found to be the major site for the stimulatory response of prostasin gene expression to SREBP-2. CONCLUSIONS: Prostasin regulates the expression of several invasion-promoting molecules in prostate cancer cells by down-modulating the EGF-EGFR signaling pathway. Active prostasin induces proteolytic cleavage in the EGFR ECD at two specific sites. One of the N-terminally truncated EGFR, the EGFR110 is auto-phosphorylated along with increased phosphorylation of downstream signaling molecules. The effect of the androgen DHT on prostasin expression in prostate cells is mediated via SREBP s, which stimulate the promoter, and Slug, which represses the promoter. Slug is up-regulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression

The epidermal growth factor receptor (EGFR) is proteolytically modified by the Matriptase–Prostasin serine protease cascade in cultured epithelial cells

AbstractProstasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase–prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling

Effects of icariin and quercetin on high glucose-induced neuronal cell apoptosis

Purpose: To study the effects of icariin and quercetin on cell apoptotic changes in neurons induced by elevated glucose condition, and the  mechanism involved. Methods: Neonatal male Sprague Dawley rats (n = 48) weighing 5 – 7 g were used. Neuronal cells were isolated from rat hippocampus and cultured after purification. The cells were randomly assigned to six groups: control, high glucose, icariin, quercetin, serine/threonine-specific protein kinase (Akt) inhibitor, and Akt agonist groups. The Akt inhibitor and agonist used in this study were MK-22062hci and SC79, respectively. The influence of icariin and quercetin on neuronal apoptotic changes were determined flow cytometrically, while their effects on levels of expression of Akt, p-Akt, bax and bcl-2 were determined with Western blotting. Results: Treatment with icariin or quercetin significantly inhibited apoptosis induced by high glucose. The concentrations of Akt, p-Akt, and bcl-2 proteins were markedly upregulated in high glucose group, relative to control (p < 0.05). The corresponding expression of bax was significantly down-regulated in high glucose group, relative to control (p < 0.05). Treatment with icariin or quercetin, or their agonists reversed high glucose-mediated alterations in these protein levels (p < 0.05). Conclusion: Icariin and quercetin inhibit neuronal cell apoptosis induced by high glucose through upregulation of bcl-2 expression and down- regulations of bax expression and Akt-induced protein phosphorylation. Thus, Icariin and quercetin possess potential benefits for the treatment of neurological diseases. Keywords: Apoptosis, High glucose condition, Hippocampal neurons, Icariin, Querceti

SoK: Play-to-Earn Projects

Play-to-earn is one of the prospective categories of decentralized applications. The play-to-earn projects combine blockchain technology with entertaining games and finance, attracting various participants. While huge amounts of capital have been poured into these projects, the new crypto niche is considered controversial, and the traditional gaming industry is hesitant to embrace blockchain technology. In addition, there is little systematic research on these projects. In this paper, we delineate play-to-earn projects in terms of economic & governance models and implementation and analyze how blockchain technology can benefit these projects by providing system robustness, transparency, composability, and decentralized governance. We begin by identifying the participants and characterizing the tokens, which are products of composability. We then summarize the roadmap and governance model to exposit there is a transition from centralized governance to decentralized governance. We also classify the implementation of the play-to-earn projects with different extents of robustness and transparency. Finally, we discuss the security & societal challenges for future research in terms of possible attacks, the economics of tokens, and governance

MEV Makes Everyone Happy under Greedy Sequencing Rule

Trading through decentralized exchanges (DEXs) has become crucial in today's blockchain ecosystem, enabling users to swap tokens efficiently and automatically. However, the capacity of miners to strategically order transactions has led to exploitative practices (e.g., front-running attacks, sandwich attacks) and gain substantial Maximal Extractable Value (MEV) for their own advantage. To mitigate such manipulation, Ferreira and Parkes recently proposed a greedy sequencing rule such that the execution price of transactions in a block moves back and forth around the starting price. Utilizing this sequencing rule makes it impossible for miners to conduct sandwich attacks, consequently mitigating the MEV problem. However, no sequencing rule can prevent miners from obtaining risk-free profits. This paper systemically studies the computation of a miner's optimal strategy for maximizing MEV under the greedy sequencing rule, where the utility of miners is measured by the overall value of their token holdings. Our results unveil a dichotomy between the no trading fee scenario, which can be optimally strategized in polynomial time, and the scenario with a constant fraction of trading fee, where finding the optimal strategy is proven NP-hard. The latter represents a significant challenge for miners seeking optimal MEV. Following the computation results, we further show a remarkable phenomenon: Miner's optimal MEV also benefits users. Precisely, in the scenarios without trading fees, when miners adopt the optimal strategy given by our algorithm, all users' transactions will be executed, and each user will receive equivalent or surpass profits compared to their expectations. This outcome provides further support for the study and design of sequencing rules in decentralized exchanges.Comment: 14 Pages, ACM CCS Workshop on Decentralized Finance and Security (DeFi'23

Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay

Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC50 values in the WT reporter assay showed near-perfect correlation (R2 = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition

Research on operation scenario based aircraft power system architecture analysis and modelling

Aircraft power system is a complex system consisting of the power generation system, the power management and distribution system, and the power consumption system, which accounts for the aircraft's major fuel consumption and emissions. This paper proposes a scenario-based comprehensive power requirement analysis and system architecture methodology in order to alleviate the risk of systems over-design and discoordination caused by traditional bottom-up load collection and individual system design. Starting from the operation scenario, system functions are identified and corresponding physical architecture and power loads are analysed. Given the complexity of operation scenarios and aircraft power system, model-based system engineering methodology is applied to the top-down aircraft power system architecture design. SysML tool is used to carry out to analyse the aircraft power system architecture during taxi scenario, which provides great advantages on model tracing and reuse

The epidermal growth factor receptor (EGFR) is proteolytically modified by the Matriptase-Prostasin serine protease cascade in cultured epithelial cells

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two aminoterminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin\u27s role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are notresponsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling. (C) 2007 Elsevier B.V All right