Identification and validation of an interaction between the E3 Ubiquitin Ligase WWP1 and the transcriptional co-activator WBP2 in the human heart

Impaired proteostasis is a cellular hallmark of aging, and the ubiquitin-proteosome system is a fundamental driver of proteostasis. As an E3 ubiquitin ligase, WW-domain containing protein 1 (WWP1) expression and activity are tightly regulated in cells, while its deregulation has been described in cancer, in neurodegenerative diseases, and in heart failure. However, the protein-protein interaction network of WWP1 is understudied, particularly in the heart. Here, we conducted a yeast-two hybrid (Y2H) screen of a human heart library and identified 21 putative WWP1 interactors, including 12 whose expression and potential function in the heart were previously unappreciated. Central in the identified protein-protein interaction network was WBP2 (WW domain binding protein 2), an oncogenic transcriptional co-activator. Utilizing immunofluorescence and proximity ligation assays, it was confirmed that endogenous WWP1 can co-localize and interact with WBP2 in human heart tissue, and, using the Y2H system, we showed that this interaction is dependent upon the associations between WW domains 1 and 3 from WWP1 and PY domains 2 and 3 of WBP2. In total, these data serve as a launching pad to identify broader protein networks regulated by WWP1 and the regions of interaction which might be targetable to reduce hallmarks of cellular aging.