Ribonucleases as cancer and viral therapeutics: Role of Coulombic interactions.

다국어 초록 (Multilingual Abstract)

Ribonuclease A (RNase A) has served as a favorite model for protein chemists for decades, used for studies on synthesis, structure, folding, and enzymology. More recently, it was discovered that several members of the RNase A superfamily have interesting biological properties, including angiogenic, antimicrobial, antiviral, and cytotoxic effects. Indeed, one homologue, Onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma.
Several requirements must be met for a ribonuclease to be cytotoxic. First, the ribonuclease must retain conformational stability at physiological temperatures. It also must have intact catalytic activity. It must not interact with the cytosolic ribonuclease inhibitor protein (RI), which binds to members of the RNase A superfamily with extremely high affinity. Finally, a cytotoxic ribonuclease must be able to bind to the cell surface and enter the cytosol of cancer cells.
This thesis focuses on the role of Coulombic interactions in two of these processes---cellular internalization and RI binding. ONC, the prototypical cytotoxic ribonuclease, is the model ribonuclease in this work. In C HAPTER 2, a systematic analysis of the role of positively charged surface residues in the cytotoxicity of ONC is described. This study supports a model in which positively charged residues on the surface of ONC influence the translocation of the protein from endosomes to the cytosol. In C HAPTER 3, a productive interaction between RI and ONC in a solution of low salt concentration is detected, providing the first direct measurement of the affinity of these proteins.
The potential use of cytotoxic ribonucleases for their antiviral properties is described in CHAPTER 4. A zymogen that has the potential to specifically kill HIV-infected cells was created from RNase A. Activation of this zymogen is dependent on the interaction between two positively charged proteins, the zymogen and HIV protease. In this case, Coulombic interactions do not favor this activation, but potentially limit it. Finally, in C HAPTER 5, several potential future directions of this work are proposed.