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. 2013 Sep;67(1):127-38.
doi: 10.1007/s12013-013-9624-6.

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

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Free PMC article

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

Carrie E Rubel et al. Cell Biochem Biophys. .
Free PMC article

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.

Figures

Figure 1
Figure 1. Schematic model representing the ubiquitin ligase/deubiquitinating enzyme screening platform
Protein is isolated from control and experimental animal tissue or cell culture samples where the expression or activity of a ubiquitin ligase or deubiquitinating enzyme of interest is manipulated to increase or decrease, dubbed a gain-of-function or loss-of-function manipulation. Isolated protein is then quantitated and incubated overnight at 4 °C with Tandem Ubiquitin Binding Entities (TUBE) or agarose control beads. Both the bound (eluate) and unbound (supernatant) fractions are collected and subjected to 2D-DIGE. Three different 2D-DIGE gels are run, each also including a pooled internal standard sample. Gel 1 compares the control sample ubiquitin enrichment to the experimental sample ubiquitin enrichment, identifying proteins whose ubiquitination is dependent upon the ubiquitin ligase of interest. The second and third gels allow comparisons of the TUBE-selected ubiquitomes within the experimental condition (Gel 2) or control condition (Gel 3) by comparing the ubiquitin-depleted supernatants from the sample incubated with TUBE to the ubiquitin-rich sample incubated with agarose control beads. The comparison on Gel 2 identifies proteins whose ubiquitination is potentially dependent upon the ubiquitin ligase of interest. The Gel 3 comparison reveals naturally occurring ubiquitinated proteins, as here, the ubiquitin ligase of interest is unperturbed. Spots are identified as “picks” by DeCyder Analysis Software based upon the determination of relative changes in intensity between the two samples and picks are aligned across all three gel comparisons to select spots for subsequent MS/MS peptide sequencing and protein identification.
Figure 2
Figure 2. MuRF1 ectopic expression and TUBE-mediated ubiquitin enrichment
Fluorescent imaging and immunoblot verified MuRF1 ectopic expression and ubiquitin enrichment prior to 2D-DIGE a Representative fluorescence micrographs of primary cardiomyocytes after 24 h of transduction with adenovirus expressing green fluorescent reporter protein alone (Ad-GFP) or in combination with Myc-tagged MuRF1 (Ad-MuRF1) at MOI of 10. b Representative immunoblots (IB) of Myc, MuRF1, and GAPDH protein levels in extracts isolated from primary cardiomyocytes transduced with Ad-GFP (−) or Ad-MuRF1 (+) adenovirus for 24 h. The red arrow indicates endogenous MuRF1, with ectopically-expressed myc-tagged MuRF1 migrating at a slightly higher molecular weight. c Representative immunoblot of total ubiquitin from TUBE enrichment in extracts isolated from primary cardiomyocytes transduced with Ad-GFP (G) or Ad-MuRF1 (M) for 24 h as performed in the 2D-DIGE MuRF1 substrate screen. Lanes 1 and 2: input samples; lanes 3 and 4: unbound TUBE supernatant collected; lanes 5 and 6: ubiquitinated protein enrichment eluted from TUBE. From 3 independent experiments we observed an average of 30 ± 14.7% increase in total ubiquitinated protein with MuRF1 ectopic expression as measured by densitometry.
Figure 3
Figure 3. 2D-DIGE gel of the TUBE-isolated ubiquitome
2D-DIGE image analysis of ubiquitin-enriched samples eluted from TUBE identified spots for mass spectrometry protein identification. Proteins eluted from TUBE incubated with protein extract from Ad-GFP or Ad-MuRF1 transduced cardiomyocytes were labeled with Cy3 and Cy5, respectively, and separated by molecular weight and isoelectric point (Cy3-GFPeluate and Cy5-MuRF1eluate, top left and bottom left, respectively). Relative changes in protein spots were calculated using the ratio of fluorescence intensity of each fluorescent channel visualized by coloring and overlaying the Cy3-GFPeluate (green) and Cy5-MuRF1eluate images (top right). The region containing the 16 spots selected for mass spectrometry identification (top right, hashed white box) was magnified and used to generate a ratio image (Cy5/Cy3) to highlight the fold-enrichement and identification of each picked spot (bottom right).
Figure 4
Figure 4. Validation of screen-identified proteins Hspd1, Tpm1, and Atp5b as substrates of MuRF1
In vitro and in vivo data demonstrate that the screen-identified proteins Hspd1, Tpm1 and Atp5b, are MuRF1 substrates. a Representative immunoblot (IB) of Hspd1 protein levels in extracts isolated from primary cardiomyocytes transduced with Ad-GFP (G) or Ad-MuRF1 (M) adenovirus for 24 h. Lane 1 and 2: input samples (light exposure, see Supplementary Fig. 2); lane 3 and 4: Ad-GFP samples eluted from either TUBE (Tu) or agarose control beads (Ag); Lane 5 and 6: Ad-MuRF1 samples eluted from TUBE (Tu) or agarose control beads (Ag). b Immunoprecipitations (IP) of Hspd1 and Tpm1 in extracts isolated from wild-type (WT) or MuRF1 transgenic (TG) mouse hearts, subsequently immunoblotted (IB) for Hspd1 or Tpm1 and ubiquitin (Ub). Lane 1 and 2: IgG control IP; lane 3 and 4: Hspd1 (top) or Tpm1 (bottom) IP; Lane 5 and 6: 10% input of extract. Red arrows indicate ubiquitin-reactive Hspd1 or Tpm1 species in MuRF1 Tg hearts (lane 4) that are not present or are of lower relative abundance in wild-type hearts (lane 3). The black arrow indicates a non-specific band also present in the IgG control IP. c In vitro ubiquitination assays for MuRF1 ubiquitination of Hspd1 and Atp5b performed in presence or absence of purified ubiquitin or MuRF1 as indicated and detected by immunoblot analysis (IB) for Hspd1 (top) or Atp5b (bottom).

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