This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Onconase, a ribonuclease (RNase) from Northern Leopard Frogs, has shown promise as a selective chemotherapeutic and is currently undergoing Phase IIIb clinical trials for malignant mesothelioma (1,2). This member of the RNase A superfamily of ribonucleases cleaves tRNA leading to cell death via apoptosis (3,4). However, treatment with Onconase has been observed to induce some renal toxicity (5). Pancreatic RNases are small highly cationic proteins found ubiquitously across species and function to degrade RNA for digestion, gene regulation, and viral immunity. Drug design research in this area has uncovered the use of other ribonucleases in this protein family as potential cancer therapeutics (6,7). Human ribonuclease (RNase 1) and the bovine homologue (RNase A) are under inquiry as chemotherapeutics as variants selectively target cancer cells while inducing less toxic renal effects as seen with Onconase (7). The mechanism of RNase into the cell consists of the steps: binding to the cell membrane, internalization, translocation out of the endosome, escaping inhibitor binding, and finally RNA cleavage leading to cell death (8). Previous research in our lab has looked at internalization of RNases among cells of varying surface groups, yet there is no definitive data that indicates how RNase variants bind onto the surface of cancerous cells and internalize (9). However, RNase cytotoxicity has been correlated to overall cationicity (10,11) and more recently the specific distribution of positive charges (8). As such binding and internalization has been linked to the negatively charged molecules on the outer membrane of the cell. Variation of the composition on cancer cell membranes compared to normal cells might target ribonucleases selectively to cancer cells (7, 12), adding to the encouraging therapeutic index observed in animal models and human trials (13,14). The surface of cancer cells frequently display changes in glycosaminoglycan profile (15), phospholipid composition (16,17) and ganglioside array (18). Specific residues of interest include heparan sulfates, charged sugar groups such as sialic acid, and lipids such as phosphotidylserine as recent research has indicated this importance by possible therapeutic targeting (19). We hypothesize that ribonuclease binding to these specific residues on the cell surface may be a factor of protein internalization and thus affect efficiency of cytotoxicity as well as selectivity of cancerous cell lines. The structure of human RNase 1 was recently determined using NMR (20). Other recent papers have also used NMR as a method to look at specific interactions between residues of proteins (21). In order to determine RNase interactions with cell plasma components, we want to similarly label RNase 1 with 15N isotope and titrate cell membrane components including but not limited to phosphatidylserine, heparan sulfate, and sialic acid. We will then look at 2D NOESY (mixing time = ms) spectra recorded on the 600 MHz NMR spectrometer as described by the methods that solved the RNase1 structure (20). Comparison of the shifts in the spectra to the structure solved in the PDB (accession number 2K11) will indicate the location and a broad degree of binding affinity of specific interactions between the RNase 1 and plasma membrane components. The results will establish how RNase1 is bound to the cell surface components and may elucidate the mechanism of internalization. We can then repeat examination using the 15N labeled RNase A and cytotoxic variants to observe a possible correlation between cell membrane interactions and RNase cytotoxicity. Also a broad range of cell membrane components can be further investigated. This may allow for developing of RNases with higher internalization due to increased binding or targeting towards cell markers that are over-expressed on cancerous cells.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-BCMB-H (40))
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University of Wisconsin Madison
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