Proteomic Study Of The Ubiquitin E3 Ligase HUWE1 Interaction With Ubiquitin And ADP-Ribose Modified Proteins

Abstract

Ubiquitination and ADP-ribosylation are important post-translational modifications (PTMs), especially in cellular response to DNA damage. HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1) is an important ubiquitin E3 ligase involved in many cellular processes. HUWE1 contains regions involved in substrate interaction and domains for PTM recognition. Specifically, HUWE1 WWE and tandem ubiquitin-binding motif (tUBM) domains interact with ADPr/iso-ADPr and ubiquitin, respectively. However, the interaction between these domains and cellular ADP-ribose and ubiquitin and potentially substrate interactions are not well investigated. Thus, this project aimed to investigate the interactome of HUWE1 WWE and tUBM domain, focusing on their ability to bind proteins modified by mono-ADP-ribose (MAR)/poly-ADP-ribose (PAR) and ubiquitin using GST pulldown assay. First, using Western blot and confocal microscopy, distinct dynamics of ubiquitination and ADP-ribosylation were observed in response to different DNA damage treatments. Second, the study demonstrated that the WWE-tUBM domain interacts with both cellular ADP-ribose and ubiquitin. Moreover, through a proteomic approach using affinity mass spectrometry, this study identified novel proteins that interact with the WWE and tUBM domains following Ultraviolet (UV) induced DNA damage. Together, the findings of this project contribute to a better understanding of how HUWE1 engages with its substrates and highlight the role of WWE and tUBM domains and their ability to recognize PTMs in mediating these interactions.