Human pancreatic ribonuclease (RNase 1) is an enzyme that can block the flow of biochemical information by catalyzing the cleavage of cellular RNA. Our intent is to use ideas and methods from enzymology, biochemistry and molecular biology, and cell biology to reveal attributes of the structure and function of RNase 1 that can lead to toxic activity against cancer cells, and to enhance that activity. During the next grant period, this intent will be achieved in four Specific Aims.
Specific Aims. In Aim 1, we shall identify specific glycans on the surface of human cells that interact with RNase 1, characterize those interactions, and assess their biological relevance.
In Aim 2, we shall develop small-molecule fluorogenic probes for two critical steps in RNase 1-mediated cytotoxicity: endocytosis and endosomal escape.
In Aim 3, we shall determine the fate of cellular RNA exposed to an RI-evasive variant of RNase 1, and whether the products of catalysis by RNase 1 are substrates for a cytosolic enzyme, RtcB.
In Aim 4, we shall produce RNase 1 with human-like glycosylation and determine the effect of the glycans on the biochemical and biological attributes of the enzyme. Significance. The results of the research proposed herein will provide a detailed biochemical understanding of the antitumoral activity of RNase 1, and could ultimately lead to new chemotherapeutic agents based on variants of an endogenous human enzyme.

Public Health Relevance

This research project is focused on the development of a new class of chemotherapeutic agents for the treatment of cancer. The agents are based on ribonuclease, which is a human enzyme that can enter human cells and catalyze the cleavage of RNA, leading to cell death. The goal of the project is to obtain fundamental insights into the relationship between the amino-acid sequence of ribonuclease, its three-dimensional structure, and its biological function, and to use those insights to create novel ribonucleases of potential therapeutic utility.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Forry, Suzanne L
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Chyan, Wen; Raines, Ronald T (2018) Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging. ACS Chem Biol 13:1810-1823
Hoang, Trish T; Tanrikulu, I Caglar; Vatland, Quinn A et al. (2018) A Human Ribonuclease Variant and ERK-Pathway Inhibitors Exhibit Highly Synergistic Toxicity for Cancer Cells. Mol Cancer Ther 17:2622-2632
Thomas, Sydney P; Hoang, Trish T; Ressler, Valerie T et al. (2018) Human angiogenin is a potent cytotoxin in the absence of ribonuclease inhibitor. RNA 24:1018-1027
Lomax, Jo E; Eller, Chelcie H; Raines, Ronald T (2017) Comparative functional analysis of ribonuclease 1 homologs: molecular insights into evolving vertebrate physiology. Biochem J 474:2219-2233
Chyan, Wen; Kilgore, Henry R; Gold, Brian et al. (2017) Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging. J Org Chem 82:4297-4304
Hoang, Trish T; Raines, Ronald T (2017) Molecular basis for the autonomous promotion of cell proliferation by angiogenin. Nucleic Acids Res 45:818-831
Hoang, Trish T; Smith, Thomas P; Raines, Ronald T (2017) A Boronic Acid Conjugate of Angiogenin that Shows ROS-Responsive Neuroprotective Activity. Angew Chem Int Ed Engl 56:2619-2622
Andersen, Kristen A; Smith, Thomas P; Lomax, Jo E et al. (2016) Boronic Acid for the Traceless Delivery of Proteins into Cells. ACS Chem Biol 11:319-23
Arnold, Ulrich; Raines, Ronald T (2016) Replacing a single atom accelerates the folding of a protein and increases its thermostability. Org Biomol Chem 14:6780-5
Thomas, Sydney P; Kim, Eunji; Kim, Jin-Soo et al. (2016) Knockout of the Ribonuclease Inhibitor Gene Leaves Human Cells Vulnerable to Secretory Ribonucleases. Biochemistry 55:6359-6362

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