Utilizing protein-ligand interactions to control biological function [electronic resource]

The ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. Chemical approaches to probe biological function have greatly contributed to the understanding of protein functions. While small-molecule inhibitors offer rapid and reversible control of protein functions, identification and development of specific inhibitors for every protein of interest remains a challenge. In the past decade, numerous technologies have been developed that combine genetic with chemical methods to create conditional protein control systems with impeccable specificity. These systems include inducible protein localization using chemical inducer of dimerization, such as rapamycin, and inducible protein stabilization system such as our destabilizing domain (DD) technology previously developed by L. Banaszynski. Highly specific, high-affinity protein-ligand interactions are key to their effectiveness. In this thesis, three technologies that utilize highly specific protein-ligand interactions are discussed. The first chapter of this thesis focuses on the development of a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of E. coli dihydrofolate reductase (ecDHFR) were engineered to have ligand-dependent stability, and when this destabilizing domain is fused to a protein of interest, the instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilized the destabilizing domain in a rapid, reversible and dose-dependent manner, and protein levels in the absence of TMP were barely detectable. The ability of TMP to cross the blood-brain barrier enabled the tunable regulation of YFP expressed rat striatum. The second chapter of this thesis describes the development of a technique in which a protein of interest is degraded in the presence of a ligand. In this system, we regulated the stability of a receptor protein of an E3 ligase complex using the previously developed destabilizing domains. A DD-fused receptor protein cannot recruit the substrate to the E3 ubiquitin ligase in the absence of ligand. Upon addition of ligand, the receptor protein is stabilized and can successfully promote ubiquitination and degradation of the substrate protein. We used HIV-1 Vif protein, a receptor protein of the Cul5 E3 ligase complex, and its substrate, human APOBEC3G. We were able to induce degradation of GFP fused APOBEC3G upon addition of ligand. Degradation of GFP occurred rapidly, tunably, and reversibly. The advantage of this system over the DD technology is that it does not require continuous administration of the ligand until the desired experimental window and is thus better suited for in vivo applications. By limiting the dosage to only during the knockout window, the cost of dosing is dramatically decreased and side effects from long-term administration of ligand can be minimized. The third and last chapter of this thesis describes an attempt to develop a new approach to induce genome modification at a specific site with high efficiency in mammalian cell lines. While there are several successful nuclease-based gene-targeting approaches that exist today, these technologies require extensive engineering and screening to isolate efficient and specific nucleases that bind to the target sites. Our strategy was to simplify the design of DNA targeting domains by using an oligonucleotide analogue, peptide nucleic acid (PNA). PNAs incorporate DNA bases on peptide backbones and make base-specific contacts with the target DNA site. The PNA domain is coupled to TMP, which then allows recruitment of the nuclease domain fused to ecDHFR. The nuclease domain is made up of a single-chain, pseudohomodimer FokI catalytic domain that non-specifically cleaves the DNA. We could not produce any recombination activity in cell. However, in vivo experiments revealed successful target DNA binding by the PNA, as well as TMP-PNA/ecDHFR-FokI binding.

Keywords: Chemistry
Advisor: Wandless Thomas primary advisor. Thesis advisor
Khosla Chaitan
Kool Eric T. advisor. Thesis advisor