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“Lift and Separate” Urokinase Plasminogen Activator Receptor (uPAR) and Its Role in Metastasis Nathan Hale High School SMART Team: Karmenleen Bajwa, Ashley Brost, Nick Gonzalez, Sukhwinder Kaur, Callan Loberg, Joelle Pietrzak, Rachel Pietrzak, Jamie Rypel, Samantha Toth, Kyle Tretow Teachers: Susan Getzel and Anne Xiong Mentor: Nancy M. Dahms, Ph.D., Dept. of Biochemistry, Medical College of Wisconsin Abstract Cancer is spread by the plasminogen activation system which is also responsible for biological processes including clearance of fibrin clots, cell migration, and activation of growth factors. The key role is played by urokinase plasminogen activator receptor (uPAR) which is a tethered membrane protein receptor having three domains, one of which is critical in activating its substrate, the serine protease urokinase plasminogen activator (uPA). uPA activation begins when two of uPA’s domains (an N-terminal growth factor domain (GFD) and a kringle domain) interact with domain one of uPAR, creating a tight bond which converts uPA to its active form. The proteolytic cascade reaction continues when activated uPA converts inactive plasminogen to the active protease plasmin. Plasmin is a multi-use protease that can activate several matrix metalloproteinases, which along with plasmin, leads to digestion of extracellular matrix (ECM) and enhanced cellular migration. The binding of uPA to uPAR localizes these proteolytic cascades to the migrating edge of the cell, thereby clearing a path in the extracellular matrix that the cells can move through. Tumor cells often express high levels of uPA and uPAR, facilitating metastasis. uPA-uPAR expression can change a benign tumor into a malignant tumor. The activity of uPAR can be regulated by the proteolytic removal of its Nterminal D1 domain. When uPAR’s N-terminal D1 domain is disabled or removed it cannot bind to uPA, therefore the cancer cells lack the ability to metastasize. This prevention technique could lead to the cure for cancer. Background A number of factors can cause tumors, including environmental carcinogens and inherited genetic mutations. Tumors are classified as either benign or malignant. Malignant cells have the ability to spread throughout the body by detaching from the tumor and entering the circulatory system. When cells in a malignant tumor invade other body tissues it is referred to as cancer cell metastasis. The cancer cells deprive other cells of nutrients. Therefore, the cells cannot function and this will eventually lead to cell death. Cancer is the result of these processes. Urokinase plasminogen activator receptor (uPAR) which is overexpressed in cancer cells, allows a benign tumor to become malignant by taking part in the digestion of the extracellular matrix (ECM). Once the ECM is gone, cells are free to move. Exploration of uPAR’s binding site may help prevent cancer metastasis. Deterioration of Extracellular Matrix (ECM) in Tumor Cells Leads to Cancer Cell Metastasis ECM Degradation Intact ECM B. Plasmin Mr x 10-3 A: uPA is activated after binding to uPAR. 1 2 3 9467- uPA and uPAR 43- The activation of uPA requires the binding of uPAR with domain 1 (D1), in order for uPA to activate plasminogen. The activation of plasminogen leads to the activation of plasmin, which degrades the ECM. The inactivation of uPA can be achieved by cleaving D1 from uPAR. Active: uPA (gray) bound to uPAR, mainly through interactions between the hydrophobic amino acid residues (yellow). uPAR with all three domains connected. The binding crevice for uPA can be seen where the amino acids are highlighted (yellow and chartreuse). Inactive: uPAR with domain 1 (orange) cleaved by protease, at the linker region, from domain 2 (magenta) and domain 3 (green). 30- uPA and D1 2014- uPA Lane 1 is the control showing only uPA. Lane 2 is uPA bound to uPAR. In lane 3 the ~80 kDa sample is uPA with full length uPAR only. The ~30 kDa sample in lane 3 is uPA bound to only domain 1 (D1) of uPAR. Lane 3 shows that uPA must bind to domain 1 in order for protein interaction to occur. The amino acid residues highlighted in yellow represent a hydrophobic area on both D1 (orange) and uPA (gray), which leads to high binding affinity between the two. Conclusion 2fd6.pdb ECM Absent 2fd6.pdb Metastasis of a Tumor C. Cascade Reaction A. SDS-Page Gel Separation of D1 from uPAR The Interaction of uPA and uPAR Plasminogen Nathan Hale 2fd6.pdb Cancer is spread by the plasminogen activation process which is also responsible for biological functions including clearance of fibrin clots, cell migration, and activation of growth factors. When uPAR’s N-terminal D1 domain is disabled or removed it cannot bind to uPA, therefore the cancer cells lack the ability to metastasize. Applying this knowledge could lead to the prevention or treatment of cancer. References Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., & Dan, K. (1990) The Ligand-binding Domain of th e Cell Surface Receptor for Urokinase-type Plasminogen Activator. The Journal of Biological Chemistry, 7842-7847. B: Proteolytic C: Disconnected cascade reaction cells are free to begins digestion of metastasize. ECM Huai, Q., Mazar, A. P., Kuo, A., Parry, G. C., Shaw, D. E., Callahan, J., & ... Huang, M. (2006). Structure of Human Urokinase Plasminogen Activator in Complex with Its Receptor. Science, 311(5761), 656-659. Retrieved from EBSCOhost. Tumor in situ Angiogenesis begins Metastasis SMART Teams are supported by the National Institutes of Health (NIH)- National Center for Research Resources-Science Education Partnership Award (NCRR-SEPA), and an NIH CTSA Award to the Medical College of Wisconsin.