A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

Abstract

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-e-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.ubiquitin-binding protein | ubiquitin ligase | ubiquitination

Ubiquitination of exposed glycoproteins by SCF FBXO27 directs damaged lysosomes for autophagy

“…We analyzed proteins ubiquitinated upon lysosomal damage by using the trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) method, which we recently developed, to identify ubiquitin ligase substrates (18), and found that SCF FBXO27 ubiquitinates luminal sites on the lysosomal protein LAMP2.…”

Proteomic techniques to probe the ubiquitin landscape

“…After tryptic digest another level of enrichment was achieved by using a-GlyGly-Lys antibodies to pull-out ubiquitinated peptides, which were identified by MS. This identified eight ubiquitination sites in substrates of Skp2 and another six in substrates of FBXO21 [114].…”

“…By combining several UBA domains in one TUBE the specificity of the chain-enrichment can be modulated [112,113]. The technique has been applied to study specific E3 ligase substrates in vivo by overexpressing them together with a FLAG-tagged, trypsin-resistant TUBE (TR-TUBE) [114]. An interesting alternative for monitoring ubiquitin chains in vivo has Probing the ubiquitin landscape been developed by combining the TUBE-probe with fluorescent probes, allowing imaging of ubiquitin chains in certain parts of the cell [115].…”

“…An interesting alternative for monitoring ubiquitin chains in vivo has Probing the ubiquitin landscape been developed by combining the TUBE-probe with fluorescent probes, allowing imaging of ubiquitin chains in certain parts of the cell [115]. In their recent study, Yoshida et al [114] aimed to identify substrates of the ubiquitin ligase FBXO21. To achieve this, the E3 was overexpressed followed by enrichment using FLAG-tagged TR-TUBES, which -according to the authors -protected against deubiquitination.…”

Efficient monitoring of protein ubiquitylation levels using TUBEs‐based microarrays

“…Including a GST-tag on the TUBEs allows conventional affinity purification with glutathione-coupled beads. Therefore, TUBEs have been used as 'ubiquitin-traps' to isolate endogenous ubiquitylated proteins from different biological sources such as cell lines, tissues, organs [15] or to study global ubiquitylation events by mass spectrometry [16][17][18][19][20][21]. Besides the enrichment of ubiquitylated proteins on a large scale, the TUBEs affinity purification can also isolate ubiquitin-interacting partners that regulate and/ or connect with distinct functions [17,20,21].…”