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, cell biology, and molecular genetics to reveal attributes of the structure and function of RNase 1 that can lead to toxic activity against cancer cells, to enhance that activity, and to reveal fundamental information on the role of RNase 1 in mammalian biology. During the next grant period, this intent will be achieved in five 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.
In Aim 5, we shall create mice in which the gene that encodes the mouse homolog of RNase 1 has been disrupted in a conditional manner. We shall characterize the phenotype of these mice so as to reveal the role(s) of RNase 1 in a mammal. Significance: The results of the research proposed herein will provide a detailed biochemical understanding of the antitumor 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA073808-20
Application #
9185947
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Forry, Suzanne L
Project Start
1997-07-10
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
20
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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
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
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
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
Porter, Douglas F; Koh, Yvonne Y; VanVeller, Brett et al. (2015) Target selection by natural and redesigned PUF proteins. Proc Natl Acad Sci U S A 112:15868-73
Desai, Kevin K; Beltrame, Amanda L; Raines, Ronald T (2015) Coevolution of RtcB and Archease created a multiple-turnover RNA ligase. RNA 21:1866-72
Tonelli, Marco; Eller, Chelcie H; Singarapu, Kiran K et al. (2015) Assignments of RNase A by ADAPT-NMR and enhancer. Biomol NMR Assign 9:81-8

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