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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA073808-17
Application #
8631584
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
2014-01-13
Budget End
2014-11-30
Support Year
17
Fiscal Year
2014
Total Cost
$291,294
Indirect Cost
$90,934
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Hoang, Trish T; Raines, Ronald T (2016) Molecular basis for the autonomous promotion of cell proliferation by angiogenin. Nucleic Acids Res :
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
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
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
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
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
Eller, Chelcie H; Chao, Tzu-Yuan; Singarapu, Kiran K et al. (2015) Human Cancer Antigen Globo H Is a Cell-Surface Ligand for Human Ribonuclease 1. ACS Cent Sci 1:181-190
Lavis, Luke D; Raines, Ronald T (2014) Bright building blocks for chemical biology. ACS Chem Biol 9:855-66
Desai, Kevin K; Cheng, Chin L; Bingman, Craig A et al. (2014) A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation. Nucleic Acids Res 42:3931-42

Showing the most recent 10 out of 93 publications