title: 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 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.
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7126, USA.
authors.forename: Carrie E
authors.initials: CE
authors.lastname: Rubel
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Schisler
authors.forename: Eric D
authors.initials: ED
authors.lastname: Hamlett
authors.forename: Robert M
authors.initials: RM
authors.lastname: DeKroon
authors.forename: Mathias
authors.initials: M
authors.lastname: Gautel
authors.forename: Oscar
authors.initials: O
authors.lastname: Alzate
authors.forename: Cam
authors.initials: C
authors.lastname: Patterson
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D024101:Mitochondrial Proteins / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D020543:Proteome / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014162:Transfection; D000070584:Tripartite Motif Proteins; D014335:Tropomyosin / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; C557067:Hspd1 protein, rat; D024101:Mitochondrial Proteins; D009124:Muscle Proteins; D020543:Proteome; C490773:Tpm1 protein, rat; D000070584:Tripartite Motif Proteins; D014335:Tropomyosin; D025801:Ubiquitin; C499086:Trim63 protein, rat; D044767:Ubiquitin-Protein Ligases; D025261:Mitochondrial Proton-Translocating ATPases