Lauren Mayse

Date of Award

8-4-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Liviu Movileanu

Keywords

Biosensors;Nanopores;Protein hub

Abstract

As scientific advancements enhance our comprehension of the human proteome, it has become evident that protein hubs possess the potential to transform our understanding and treatment of numerous diseases. This is attributed to their multitude of binding partners, often with multiple binding sites, and their extensive range of cellular functions. Protein hubs can serve as transcriptional cofactors, facilitate the assembly of large multi-subunit complexes, and play a vital role in cellular signaling. One prime example of these multifaceted molecules is the WD40 repeat protein 5 (WDR5). This protein is crucial in the progression of various cancers. It mediates the development of Mixed Lineage Leukemia and acts as a cofactor for the oncogenic transcription factor c-Myelocytomatosis (MYC). This work centers around providing a highly detailed understanding of multiple WDR5 interactions and novel tools able to study this protein beyond current limitations. These two goals were achieved via the implementation of nanopore-based biosensor design and single-molecule electrophysiology. This protein engineering strategy created a platform far more sensitive than common commercially available instrumentation. Through this intricate and sequential series of projects, a complex interplay was uncovered between WDR5 and its binding partner, the Mixed Lineage Leukemia 4 (MLL4) enzyme, showcasing their multi-event interaction dynamics. This interaction mechanism was completely unknown previously. The work also showed a very weak but crucial interaction between WDR5-MYC. This was the first time this interaction was probed with single-molecule resolution. These nanopore-based sensors provided a new collection of information to improve our understanding of WDR5’s binding dynamics at two catalytic sites i.e. WDR5 interacting (WIN) and WDR5 binding motif (WBM) sites. This will significantly impact the strategies employed by drug developers as they endeavor to target this crucial nuclear protein hub. These studies also sparked an investigation on how to make nanopore-based sensor more available and feasible for the scientific community. In order to achieve this, there is a need for an additional approach to optimize the design of these sensors. Therefore, the final project involved integrating these nanopore sensors with biolayer interferometry (BLI) via nanodisc technology. This way, we created a high-throughput approach capable of evaluating the performance of these sensors and drastically reduced the optimization time. The hope was that this original approach would facilitate the development of more nanopore-based sensors. This work provided a better understanding of nanopore-based sensor design and WDR5 interactions.

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Open Access

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