Integrating Nature's Covalent Strategies with Proteomics to Interrogate the Function of Protein Modifications

Abstract

Post-translational modifications (PTMs) expand biological functions beyond genetic coding. This thesis harnesses nature-inspired covalent chemistries to study electrophilic protein modifications and their crosstalk with enzymatic PTMs. Mass spectrometry (MS)-based proteomics uncovers PTM functions and effects on protein-protein interactions (Chapter 1). Chapter 2 presents a chemically induced proximity approach using HaloTag technology—HaloTAC— to induce targeted protein modifications. First-generation HaloTACs recruited AMPK, ULK1, and p300/CBP to phosphorylate and acetylate model proteins Akt and PTEN. Triplex SILAC-MS revealed modification sites. Chapter 3 introduces thrombosis, a major cause of stroke and heart attack, exploring antithrombotic therapies and the link between PTM dysregulation and thrombosis. It introduces thrombosis and hemostasis pathways foundational to drug discovery. Chapter 4 identifies safer antiplatelet candidates and platelet protein targets for stroke treatment. Sulforaphane (SFN), an electrophilic natural product, showed antiplatelet activity. Activity-based protein profiling (ABPP) and label-free quantification (LFQ) proteomics identified PDIA6 as SFN’s main target, with covalent cysteine modification linked to SFN’s favorable safety profile. SFN’s stroke efficacy was confirmed in a murine model. Chapter 5 examines five electrophilic natural products (SFN, 6-shogaol, xanthohumol, 9-nitrooleic acid, and oleocanthal) and their interactions with the platelet ubiquitin-proteasome system (UPS). Competitive ABPP approaches revealed UPS-related targets, including deubiquitinases (DUBs), E1, E2, and E3 ligases, whose activities were affected by natural product binding. Two DUB-targeting chimeras (DUBTACs) were synthesized to modulate HDAC6 expression. In summary, this thesis leverages natural covalent chemistries and MS-based proteomics to study PTMs, uncover new druggable targets, and inform cardiovascular therapeutics.