Soluble urokinase plasminogen activator receptor (suPAR) in bronchoalveolar fluid and blood in critically ill patients-a prospective cohort study.

INTRODUCTION: Soluble urokinase plasminogen activator receptor (suPAR) is a biologically active protein and increased levels are associated with worse outcomes in critically ill patients. suPAR in bronchoalveolar fluid (BALF) may be helpful to differentiate between types of acute respiratory distress syndrome (ARDS) and may have potential for early detection of fungal infection. METHODS: We prospectively investigated levels of suPAR in BALF and serum in critically ill patients who underwent bronchoscopy for any reason at the ICU of the Department of Internal Medicine, Medical University of Graz, Graz, Austria. RESULTS: Seventy-five patients were available for analyses. Median age was 60 [25th-75th percentile: 50-69] years, 27% were female, and median SOFA score was 12 [11-14] points. Serum suPAR levels were significantly associated with ICU mortality in univariable logistic regression analysis. There was no correlation between BALF and serum suPAR. Serum suPAR was higher in ARDS patients at 11.2 [8.0-17.2] ng/mL compared to those without ARDS at 7.1 [3.7-10.1] (p < 0.001). BALF-suPAR was significantly higher in patients with evidence of fungal lung infection compared to patients without fungal infection both in the general cohort (7.6 [3.2-9.4] vs 2.5 [1.1-5.3], p = 0.013) and in the subgroup of ARDS (7.2 [3.1-39.2] vs 2.5 [1.0-5.2], p = 0.022). All patients were classified as putative/probable invasive aspergillosis. CONCLUSION: We found significant higher levels of serum suPAR in ARDS patients compared to those not fulfilling ARDS criteria. Serum and BALF-suPAR were significantly higher in those patients with evidence for invasive pulmonary aspergillosis. These findings may suggest testing this biomarker for early diagnosis of fungal infection in a greater cohort.

Piptide Chemotypes for Perturbation of the Interaction of Urokinase with Its Receptor.

Only a few small molecules that disrupt the uPA and uPA receptor (uPAR) interaction have been discovered despite decades of research in the area, and none have been approved in clinical trials. Research reported here features two new ways of considering the problem of discovering small molecules to disrupt uPA•uPAR, specifically in terms of chemotype design and method of evaluation. Chemotypes used in this work are piptides (Arancillo . Angew. Chem., Int. Ed., 2021, 60, 6653-6659) with side chains corresponding to the uPA loop that binds uPAR. Further, hybrids of 1 and another uPAR ligand developed in these labs (2), i.e., 3 and 4, were also designed and tested. All the piptide chemotypes bound uPAR at concentrations of 50 µM or less. Members of this series had Ki values <3 µM and showed favorable responses in cellular assays; these data are comparable with the best small molecule uPA•uPAR disruptors in the literature (from conventional screening).

Plasminogen activator receptor assemblies in cell signaling, innate immunity, and inflammation.

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are serine proteases and major activators of fibrinolysis in mammalian systems. Because fibrinolysis is an essential component of the response to tissue injury, diverse cells, including cells that participate in the response to injury, have evolved receptor systems to detect tPA and uPA and initiate appropriate cell-signaling responses. Formation of functional receptor systems for the plasminogen activators requires assembly of diverse plasma membrane proteins, including but not limited to: the urokinase receptor (uPAR); integrins; N-formyl peptide receptor-2 (FPR2), receptor tyrosine kinases (RTKs), the N-methyl-d-aspartate receptor (NMDA-R), and low-density lipoprotein receptor-related protein-1 (LRP1). The cell-signaling responses elicited by tPA and uPA impact diverse aspects of cell physiology. This review describes rapidly evolving knowledge regarding the structure and function of plasminogen activator receptor assemblies. How these receptor assemblies regulate innate immunity and inflammation is then considered.

The Urokinase Receptor: A Multifunctional Receptor in Cancer Cell Biology. Therapeutic Implications.

Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.

Crystal Structures of Human C4.4A Reveal the Unique Association of Ly6/uPAR/α-neurotoxin Domain.

Ly6/uPAR/α-neurotoxin domain (LU-domain) is characterized by the presence of 4-5 disulfide bonds and three flexible loops that extend from a core stacked by several conversed disulfide bonds (thus also named three-fingered protein domain). This highly structurally stable protein domain is typically a protein-binder at extracellular space. Most LU proteins contain only single LU-domain as represented by Ly6 proteins in immunology and α-neurotoxins in snake venom. For Ly6 proteins, many are expressed in specific cell lineages and in differentiation stages, and are used as markers. In this study, we report the crystal structures of the two LU-domains of human C4.4A alone and its complex with a Fab fragment of a monoclonal anti-C4.4A antibody. Interestingly, both structures showed that C4.4A forms a very compact globule with two LU-domain packed face to face. This is in contrast to the flexible nature of most LU-domain-containing proteins in mammals. The Fab combining site of C4.4A involves both LU-domains, and appears to be the binding site for AGR2, a reported ligand of C4.4A. This work reports the first structure that contain two LU-domains and provides insights on how LU-domains fold into a compact protein and interacts with ligands.

The urokinase plasminogen activator binding to its receptor: a quantum biochemistry description within an in/homogeneous dielectric function framework with application to uPA-uPAR peptide inhibitors.

Despite being recognized as a therapeutic target in the processes of cancer cell proliferation and metastasis for over 50 years, the interaction of the urokinase plasminogen activator uPA with its receptor uPAR still needs an improved understanding. High resolution crystallographic data (PDB ) of the uPA-uPAR binding geometry was used to perform quantum biochemistry computations within the density functional theory (DFT) framework. A divide to conquer methodology considering a mixed homogeneous/inhomogeneous dielectric model and explicitly taking water molecules into account was employed to obtain a large set of uPA-uPAR residue-residue interaction energies. In order of importance, not only were Phe25 > Tyr24 > Trp30 > Ile28 shown to be the most relevant uPA residues binding it to uPAR, but the residues Lys98 > His87 > Gln40 > Asn22 > Lys23 > Val20 also had significant interaction energies, which helps to explain published experimental mutational data. Furthermore, the results obtained with the uPA-uPAR in/homogeneous dielectric function show that a high dielectric constant value ε = 40 is adequate to take into account the electrostatic environment at the interface between the proteins, while using a smaller value of ε (<10) leads to an overestimation of the uPA-uPAR binding energy. Hot spots of the uPA-uPAR binding domain were identified and a quantum biochemistry description of the uPAR blockers uPA21-30 and cyclo21,29uPA21-29[(S21C;H29C)] was performed, demonstrating that cyclization improves the stability of mimetic peptides without compromising their binding energies to uPAR.

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.

The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus-host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus-host interaction and viral pathogenesis.

Structure-function relationship of an Urokinase Receptor-derived peptide which inhibits the Formyl Peptide Receptor type 1 activity.

The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature (i.e. the series of contacts that transmit the conformational transition throughout the complex), translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration.

Analytical, biochemical and clearance considerations of soluble urokinase plasminogen activator receptor (suPAR) in healthy individuals.

BACKGROUND: Soluble urokinase plasminogen activator receptor (suPAR) is an emerging marker of cardiovascular disease burden. Appropriate assessment of assay performance and reference interval are required to enable interpretation of results to facilitate its clinical application. METHODS: suPAR was measured using the suPARnostic® ELISA in 155 healthy volunteers. Assay performance was assessed for anticoagulant effect, recovery, interference, linearity and cross-reactivity. The identity of immunoreactive suPAR was confirmed by size-exclusion HPLC. To establish anatomical sites of release and uptake, we measured suPAR in regional samples from subjects undergoing cardiac catheterization. RESULTS: The median concentration of suPAR was 2.1 ng/mL (IQR:1.7-2.3) in health. In comparison with EDTA, suPAR measurements were affected by lithium heparin (>10% change) and increased with serum usage. suPAR reactivity also increased in the presence of haemolysis (10 g/L), but was suppressed with urokinase and lipids (4 g/L). In multiple regression analyses, suPAR associated independently with body weight, NT-proBNP and MR-proADM (P = .03) for healthy individuals. Regional plasma sampling showed lower suPAR concentrations in the coronary sinus and renal vein compared with concentrations in femoral arterial samples. Immunoreactive circulating suPAR species had Mr of 10-39 kDa. CONCLUSION: The suPARnostic® assay performs acceptably for a clinical assay but is limited in the presence of high levels of hemolysis, lipids and urokinase. We provide the first evidence for the heart and kidneys as organs of suPAR clearance in humans. Additional investigations are warranted to determine whether there is a need to compare the marker performance of differing circulating forms of suPAR.

Crystal structure of the unoccupied murine urokinase-type plasminogen activator receptor (uPAR) reveals a tightly packed DII-DIII unit.

The urokinase-type plasminogen activator receptor (uPAR) is a cell surface receptor that is capable of binding to a range of extracellular proteins and triggering a series of proteolytic and signaling events. Previous structural studies of uPAR with its ligands uPA and vitronectin revealed that its three domains (DI, DII, and DIII) form a large hydrophobic cavity to accommodate uPA. In the present study, the structure of unoccupied murine uPAR (muPAR) is determined. The structure of DII and DIII of muPAR is well defined and forms a compact globular unit, while DI could not be traced. Molecular dynamic simulations further confirm the rigid binding interface between DII and DIII. This study shows overall structural flexibility of uPAR in the absence of uPA.

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding ß3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin ß3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for ß3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

Chemical Space Overlap with Critical Protein-Protein Interface Residues in Commercial and Specialized Small-Molecule Libraries.

There is growing interest in the use of structure-based virtual screening to identify small molecules that inhibit challenging protein-protein interactions (PPIs). In this study, we investigated how effectively chemical library members docked at the PPI interface mimic the position of critical side-chain residues known as "hot spots". Three compound collections were considered, a commercially available screening collection (ChemDiv), a collection of diversity-oriented synthesis (DOS) compounds that contains natural-product-like small molecules, and a library constructed using established reactions (the "screenable chemical universe based on intuitive data organization", SCUBIDOO). Three different tight PPIs for which hot-spot residues have been identified were selected for analysis: uPAR⋅uPA, TEAD4⋅Yap1, and CaV α⋅CaV ß. Analysis of library physicochemical properties was followed by docking to the PPI receptors. A pharmacophore method was used to measure overlap between small-molecule substituents and hot-spot side chains. Fragment-like conformationally restricted small molecules showed better hot-spot overlap for interfaces with well-defined pockets such as uPAR⋅uPA, whereas better overlap was observed for more complex DOS compounds in interfaces lacking a well-defined binding site such as TEAD4⋅Yap1. Virtual screening of conformationally restricted compounds targeting uPAR⋅uPA and TEAD4⋅Yap1 followed by experimental validation reinforce these findings, as the best hits were fragment-like and had few rotatable bonds for the former, while no hits were identified for the latter. Overall, such studies provide a framework for understanding PPIs in the context of additional chemical matter and new PPI definitions.

Surface-enhanced Raman spectroscopy (SERS) nanoprobes for ratiometric detection of cancer cells.

We report a ratiometric strategy for detection of different types of breast cancer cells by surface-enhanced Raman spectroscopy (SERS), which simultaneously quantifies the levels of dual biomarkers distinctly expressed on cancer cells to consequently achieve their expression ratio. Two SERS nanoprobes that are encoded with distinct SERS signatures are conjugated with urokinase plasminogen activation receptor (uPAR)- and epidermal growth factor receptor (EGFR)-targeting peptides. The SERS imaging of single live cells can accurately quantify the cellular biomarker expression difference from the SERS intensity ratio and is further employed for cancer cell screening. The results show that MDA-MB-231 and MCF-7 exhibit distinct expression of the uPAR and EGFR and they can be respectively discriminated by the intensity ratio of SERS signals from uPAR- and EGFR-targeting SERS nanoprobes. The ratiometric strategy permits background-free SERS detection of cancer cells and dramatically improves the signal-to-noise ratio of targeted cellular SERS imaging, thus enabling accurate cancer cell screening without the need for additional references. It is believed that the present ratiometric method should be a promising avenue for breast cancer diagnostics and screening, which can be easily extended for detection of other cancer cell types.

TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation.

BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.

Urokinase receptor derived peptides as potent inhibitors of the formyl peptide receptor type 1-triggered cell migration.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser88-Arg-Ser-Arg-Tyr92, SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration.

D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo.

D2A-Ala is a synthetic peptide that has been created by introducing mutations in the original D2A sequence, 130IQEGEEGRPKDDR142 of human urokinase receptor (uPAR). In vitro, D2A-Ala peptide displays strong anti-tumoural properties inhibiting EGF-induced chemotaxis, invasion and proliferation of a human fibrosarcoma cell line, HT 1080, and a human colorectal adenocarcinoma cell line, HT 29. D2A-Ala exerts its effects by preventing EGF receptor (EGFR) phosphorylation. To test D2A-Ala in vivo, this peptide was PEGylated generating polyethyleneglycol (PEG)-D2A-Ala peptide. PEGylation did not alter the inhibitory properties of D2A-Ala. Human tumour xenografts in the immunodeficient nude mice using HT 1080 and HT 29 cell lines showed that PEG-D2A-Ala significantly prevents tumour growth decreasing size, weight and density of tumours. The most efficient doses of the peptide were 5 and 10 mg/kg, thereby relevant for possible development of the peptide into a drug against cancer in particular tumours expressing EGFR.

D2A sequence of the urokinase receptor induces cell growth through αvß3 integrin and EGFR.

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvß3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.

Expression and crystallographic studies of the D1D2 domains of C4.4A, a homologous protein to the urokinase receptor.

C4.4A is a glycosylphosphatidylinositol-anchored membrane protein comprised of two LU domains (Ly6/uPAR-like domains) and an extensively O-glycosylated C-terminal Ser/Thr/Pro-rich region. C4.4A is a novel biomarker for squamous epithelial differentiation. Its expression is dysregulated under various pathological conditions and it is a robust biomarker for poor prognosis in various malignant conditions such as pulmonary adenocarcinoma. To facilitate crystallization, the two LU domains were excised from intact C4.4A by limited proteolysis, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to 2.7 Šresolution and belonged to space group C2221, with unit-cell parameters a = 55.49, b = 119.63, c = 168.54 Å. The statistics indicated good quality of the data, which form a solid basis for the determination of the C4.4A structure.