The
ubiquitin-
proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as
cancer, muscle-wasting, and neurodegeneration.
Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of
transcription factors and other signaling molecules. Substrate-specificity is a critical regulatory step of ubiquitination and is mediated by
ubiquitin ligases. Recent studies implicate
ubiquitin ligases in multiple models of
cardiac diseases such as
cardiac hypertrophy,
atrophy, and
ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac
ubiquitin ligases provides both mechanistic insights into
heart disease as well as possible therapeutic targets. Current methods identifying substrates for
ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying
ubiquitin ligase substrates utilizing tandem
ubiquitin binding entities technology, two-dimensional differential in gel electrophoresis, and mass spectrometry, validated by the identification of both known and novel physiological substrates of the
ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any
ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various
biological conditions, opening the door to a clearer mechanistic understanding of
ubiquitin ligase function and broadening their potential as therapeutic targets.