Molecular mechanisms underlying regulation within the ubiquitin proteasome pathway

Abstract

Tight regulation of protein homeostasis is critical for maintaining cellular processes. The ubiquitin proteasome pathway (UPP), one of the core regulatory pathways within the cell, acts globally to orchestrates protein stability. Ubiquitin is a finely conserved, 76 amino acid protein, featuring a β-grasp fold with a six amino acid tail at its C-terminus. The covalent transfer of ubiquitin, a 76-amino acid (aa) post-translational modification, to a protein target is a tightly regulated process, enacted through a three-part cascade. First, ubiquitin activation by the E1 enzyme (n = 2, humans) results in ubiquitin thioester bond to a ubiquitin-conjugating E2 enzyme (n > 50, humans) in an ATP-dependent fashion. Following, the formation of ubiquitin-protein conjugates is catalyzed by E3 ubiquitin ligases (n > 600, humans). Substrate specificity of ubiquitin transfer is regulated by the diversity of E3 ubiquitin ligases, whereas specificity of ubiquitin transfer is regulated jointly by the E2 and E3 enzymes. Collectively, the ubiquitin proteasome pathway forms a reversible, interconnected quality control network self-organized through biophysical properties and subcellular compartmentalization. A fundamental question is how substrate is selectively targeted for proteolysis. In part one of this thesis, through structural and biochemical techniques, we explore how a prototypical ubiquitin ligase uses a cryptic ubiquitin binding site to constrain activity and confer chain selectivity within its distal catalytic domain. We leverage cryogenic electron microscopy to determine an atomic model of the apo-ligase and low energy intermediates associate with ubiquitin transfer. We demonstrate that the ligase architecture and domain motions of the noncatalytic modules is finely conserved between S. cerevisiae ortholog Tom1 and human ortholog HUWE1. Our model sheds light on how patient mutations distal to the catalytic module could influence activity of the human HUWE1.