Regulation Of Apobec3B Via Interacting Proteins

Abstract

Proteins play a major role in cellular processes; therefore, it is important to understand how they perform their biological functions. However, proteins rarely act independently, but rather they often create “molecular machines” and have intricate physical and chemical dynamic interactions to undertake biological functions at both cellular and system levels. A key step towards unraveling the complex molecular relationships in living systems lies in the mapping and characterization of protein-protein interactions (PPIs). These interactions form the backbone of signal transduction pathways and cellular networks in diverse physiological processes. Due to their critical roles in relaying key cellular signals in both normal and cancer cells; once deemed “undruggable” PPIs have led to the development of a potential new class of drug targets [for example, the interaction between p53 and MDM2 (1)]. In this thesis, we try to understand the interactome of APOBEC3B (A3B), a key molecular driver inducing mutations in multiple human cancers. We utilized an affinity purification mass spectrometry approach to identify cellular proteins that interact with A3B. Our results revealed a specific interaction with the cell cycle protein cyclin-dependent kinase 4 (CDK4). We validated and mapped this interaction by structure guided mutagenesis and co-immunoprecipitation experiments. Functional studies and immunofluorescence microscopy experiments in multiple cell lines revealed that A3B is not a substrate for CDK4-Cyclin D1 phosphorylation, nor is its deaminase activity modulated. Instead, we found that A3B is capable of disrupting the CDK4-dependent nuclear import of Cyclin D1. We propose that this interaction may favor a more potent antiviral response and simultaneously facilitate cancer mutagenesis. In addition, our studies on the A3B “interactome” led to multiple advancements in genome engineering, specifically in cytosine base-editing (CBE). With the recent discovery of base editing systems in which a DNA editing enzyme, such as an APOBEC, is targeted to a specific base by covalent fusion to a Cas9 nickase (Cas9n) complex the prospect of precise genome engineering has become reality. However, current CBEs are prone to undesirable off-target mutations (most frequently occurring as target-adjacent mutations). Through the identification of novel A3B interacting proteins we developed a method, termed “MagnEdit”, in which we use these interacting proteins to serve as “magnets” fused to Cas9n in order to attract A3B to single-base editing hotspots. By untethering A3B from Cas9n and utilizing endogenous A3B “recruiters” we have dramatically reduced off target editing. These findings, while demonstrated here for A3B and its interacting proteins, have implications for other editing technologies and other recruiters such as antibodies, small molecules, and epitope tags.