"Chemical tools and strategies to engineer and discover new biology"
My dissertation focuses on using chemical tools and insight to engineer and discover new biology. Two themes are covered herein: (1) developing a modular engineering platform for intracellular delivery of protein cargo to erythroid lineage cells, and (2) establishing methods to unravel sterol lipid interactome of the bacterium Enhygromyxa salina to discover novel sterol roles for the lipid in bacteria. In Part 1, I describe the development of a modular protein engineering platform that enables the “plug and play” design of the first cell-permeable nanobody degrader of BCL11A. This degrader is capable of robust de-repression of fetal hemoglobin in human umbilical cord blood-derived erythroid progenitor cells (HUDEP-2) and primary hematopoietic stem (CD34+). We followed this with designs that improved on the low efficiency of delivery and lack of cell-type specificity. To accomplish this, we evaluated the performance of various protein-based delivery vehicles such as cell-penetrating peptides (CPPs), and thiol-reactive additives, among others, for their ability to deliver cargo to erythroid precursor cells. We then used receptor targeting to engineer selectivity to erythrocytes over other cell types. Together, these findings advance our understanding of protein delivery to challenging cell types and illustrate some of the intricacies of cell-surface receptor targeting. In part two, I describe our efforts to unravel the biochemical and molecular mechanisms involved in bacterial-sterol interactions in Enhygromyxa Salina. E. salina is the first reported bacteria capable of de novo production of cholesterol. The biosynthesis of complex sterol lipids in bacteria has implications for theories of evolution, bacteria biology, and bacterial-sterol interactions within human biomes. To elucidate the role of sterols in E. salina, we first used thermal proteome profiling (TPP) to define the putative cholesterol and desmosterol interactome, then biochemically and bioinformatically characterized two hits of high interest. The first is a putative sterol sulfotransferase that shows selective activity with desmosterol and indicates that E. salina is capable of downstream modification of complex sterols. The second is an uncharacterized protein with more than 1000 bacterial homologs, with some produced in organisms known to require or produce cholesterol. Together our work points to cholesterol and desmosterol playing unique roles in signaling, transcriptional regulation, and membrane stabilization in bacteria.
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