Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

Abstract

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 (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), 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.

Ube2g2-gp78-mediated HERP polyubiquitination is involved in ER stress recovery

“…It was reported that HERP is strongly upregulated both at the mRNA and protein level by homocysteine and other ER stress inducers (Hori et al, 2004;Kokame et al, 2000;Rubel et al, 2013). We firstly confirmed that ER stress inducers such as homocysteine, b-mercaptoethanol, tunicamycin, thapsigargin and dithiothreitol (DTT) but not oxidative ER stress inducers such as H 2 O 2 and Paraquat caused the accumulation of HERP protein in HEK293T, HeLa and HCT116 cells by immunoblotting ( Fig.…”

“…Interaction between gp78 CUE and the HERP UBL domain is essential for HERP polyubiquitylation Substrate specificity is usually determined by E3 (Pickart, 2001;Rubel et al, 2013). We therefore tested whether there was a physical interaction between gp78 and HERP.…”

Systematic approaches to identify E3 ligase substrates

“…This method does not rely on protein overexpression, tag fusion proteins, inhibitors or chemical and genetic manipulations and therefore is ideal for the detection of endogenous ubiquitylated proteins [121,122]. To identify substrates of the MuRF1 E3 ligase, TUBEs coupled with two-dimensional differential in-gel electrophoresis (2D-DIGE) and MS were used to compare cells overexpressing either MuRF1 or GFP [93]. This study identified 20 proteins with differential ubiquitylation following MuRF1 overexpression, 3 of which were previously known substrates and 3 that were validated as novel substrates.…”

Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases

“…TUBES have become a widely used tool in molecular biology to trap ubiquitin-bound substrates and are commercially available. New substrates for the striated muscle-specific RING E3 MuRF1 were identified with the use of TUBES, in combination with two-dimensional differential in gel electrophoresis (2D-DIGE) and matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry [98]. This technique centers on the comparative analysis of cells with differential (reduced or enhanced) E3 ligase activity.…”