Endogenous and chemical modifications of model proteins

Abstract

Protein modifications are ubiquitous in nature, introducing biological complexity and functional diversity. Of the known post-translational modifications, glycosylation is one of the most common and most complex, yet some of the biological implications of this modification remain poorly understood. The development of chemical tools to mimic these modifications is helping to elucidate their biological roles and improve the range of biopharmaceuticals. To probe the biochemistry of endogenous glycosylation and to test the efficacy of novel synthetic modifications, tractable protein scaffolds are needed. Previously, members of the pancreatic-type ribonuclease (ptRNases) superfamily have been utilized as model protein scaffolds. They are a class of highly conserved, secretory endoribonucleases that mediate diverse biological functions through the cleavage of RNA.

The prototypical family homolog, human ribonuclease 1 (RNase 1), has been observed as a differentially glycosylated protein in vivo and been shown to tolerate a wide range of chemical manipulations. It has also emerged as an ideal candidate for protein-based drug therapy. The goal of this thesis is to showcase the biological potential of RNase 1 as a model endogenously glycosylated protein and as a protein payload for evaluating intracellular delivery systems. In CHAPTER 1, I summarize the current knowledge about ptRNases including their biochemical characterization, conservation of N-glycosylation, and therapeutic potential. RNase 1 possesses three N-glycosylation sites giving rise to enormous heterogeneity in biological samples, with unknown implications. In CHAPTER 2, I demonstrate that glycosylation of RNase 1 enhances protein stability and attenuates enzymatic activity.

In CHAPTER 3, I utilize a previously developed diazo compound to enhance delivery of a therapeutically relevant RNase 1 variant. The modification is shown to be reversed upon entry into the cell, presenting a novel approach for delivering native, functional proteins to the cytosol. Intracellular delivery of another model protein, Cytochrome C (CytoC), has shown therapeutic promise as well. In CHAPTER 4, I demonstrate that synthetic glycosylation with a large, monofunctionalized dextran conveys CytoC into the intracellular space, triggering apoptosis. Finally, CHAPTER 5 outlines future directions for the study of RNase 1 glycosylation and expanding the utility of the established diazo and dextran-based delivery systems. Taken together, this thesis explores a wide variety of protein modifications, demonstrating biochemical effects of endogenous glycosylation and enhanced delivery of protein payloads with chemical tools.