Access Type

Open Access Dissertation

Date of Award

January 2025

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary k. Pflum

Abstract

Post-translational modifications (PTMs) are essential as chemical changes that regulate protein function, activity, stability, interactions, and localization after protein synthesis. PTMs, such as phosphorylation, acetylation, and ubiquitination, can activate or deactivate proteins, influence their interactions with other molecules, and determine their cellular location. This regulation is vital for numerous biological processes, such as cell cycle control, signal transduction, and gene expression. Abnormal PTMs are linked to various diseases in humans, including parasitic infection, blood disorders, cancers, and neurodegenerative diseases, making their study crucial for understanding disease mechanisms and developing targeted therapies. In the Pflum lab, we are interested in the PTMs in cell biology to understand the molecular basis of disease. We are mainly interested in two proteins that regulate PTMs involved in human diseases: kinase and histone deacetylase enzymes. The current gap in the field for both kinases and HDAC is the lack of tools available to identify both the protein substrates and associated proteins. Our goal is to use a chemical approach combined with LC-MS/MS to characterize the substrates of kinases and HDAC proteins in human aliments, which will lead to the identification of novel drug targeting and biomarkers to diagnose illness. To improve the gap in the field for studying kinases, the Pflum lab has developed kinase-catalyzed labeling and crosslinking techniques to identify kinase-substrate pairs. The methods utilize γ- modified ATP analogs that contain crosslinking analogies, which help identify kinase-substrate pairs by labeling substrates or stabilizing the kinase-substrate interactions through crosslinking. K-CLASP (Kinase Catalyzed Crosslinking and Streptavidin Purification) is a method for identification of phosphosite-specific kinase and unanticipated protein-protein interactions in phosphorylation-mediated biological events. K-CLASP was successfully applied to Plasmodium falciparum in two collaborative studies with Dr. Lanzer's group at Heidelberg University Hospital, which discovered kinases and protein-protein interactions. The data provided can lead to improved development of therapeutics for malaria. Lastly, a new and state-of-the-art method was created in the Pflum to study Histone deacetylase1 (HDAC1) proteins, which are viable targets for future sickle cell disease (SCD) drug development. HDAC1 inhibitors influence globin switching. Unfortunately, the limited understanding of the role of HDAC1 in sickle cell biology and globin switching has stalled efforts to develop HDAC-targeted drugs for SCD symptoms. HDAC proteins catalyze the deacetylation of acetyl-lysine residues on HDAC proteins and are epigenetic regulators of gene expression. However, their role in regulating cellular activities beyond gene expression has grown. Here, we identified HDAC1 substrates in primary erythroblasts from sickle cell patients to characterize HDAC1 activity using substrate-trapping and LC/MS/MS. A requirement of substrate trapping is the transient expression of mutant HDAC1 in the cell line of interest. Due to poor transfection efficiency, overexpressing inactive mutants of HDAC1 in primary erythroblast cells is challenging and prevents direct trapping. To overcome the transfection challenge, we developed a double immunoprecipitation (dIP) trapping method to identify substrates and characterize the activity of HDAC1 proteins in SCD. Twenty-eight proteins were enriched in three out of three trials. Out of those 28 proteins, three were selected to be further investigated for their significance to sickle cell anemia. With the molecular mechanism of HDAC1 activity in SCD known, targeted drug development will become possible.

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