Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins

Mark, Kevin G.; Simonetta, Marco; Maiolica, Alessio; Seller, Charles A.; Toczyski, David P.

doi:10.1016/j.molcel.2013.12.003

Cited by 49 publications

(73 citation statements)

References 54 publications

(61 reference statements)

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“…In that method, ligase-substrate affinity is increased by fusing the ligase to a tandem ubiquitin-associated domain (35). Although this method should also be useful for identification of the substrates of ubiquitin ligases, it requires parameters such as linker length and configuration to be optimized for each F-box protein.…”

Section: Discussionmentioning

confidence: 99%

A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

Yoshida¹,

Saeki²,

Murakami³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

104

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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

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Section: Discussionmentioning

confidence: 99%

A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

Yoshida¹,

Saeki²,

Murakami³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

104

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“…The potential to capture potentially weak and transient proteinprotein interactions is a key feature of the UBAIT approach. Other approaches for characterizing E3s have been recently reported, including one that fuses UBA ubiquitin binding domains to F-box proteins to capture the product of F-box protein-mediated ubiquitylation events [34]. While the UBA fusion approach was designed to specifically characterize ubiquitin ligases, the UBAIT approach is not necessarily limited to characterizing ubiquitin ligases.…”

Section: Discussionmentioning

confidence: 99%

“…Huibregtse, unpublished observation). Similar to the UBAIT concept, the "NEDDylator" system and the BiolD biotinylation-based proximity ligation approaches can potentially be adapted for any POI [33,34]. The NEDDylator is a POI-Ubc12 fusion protein, which can be added to cell lysates or expressed in cells along with a tagged form of NEDD8.…”

Section: Discussionmentioning

confidence: 99%

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

et al. 2015

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We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (UbiquitinActivated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1-and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ-a histone protein involved in DNA repair-as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

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“…Identifying E3-substrate pairs can be difficult given the relatively weak affinities of enzyme for substrate and the low stoi-chiometry of ubiquitinated proteins (15). To address this gap, diverse approaches have been developed in recent years including functional genomics with targeted siRNA libraries (16 -18), global protein stability profiling (19 -21), extract-based functional assays (22)(23)(24), and affinity-based proteomics strategies (20,(25)(26)(27).…”

mentioning

confidence: 99%

Quantitative Lys-ϵ-Gly-Gly (diGly) Proteomics Coupled with Inducible RNAi Reveals Ubiquitin-mediated Proteolysis of DNA Damage-inducible Transcript 4 (DDIT4) by the E3 Ligase HUWE1

Thompson

Nagel

Hoving

et al. 2014

Journal of Biological Chemistry

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Background: HUWE1 is an E3 ligase implicated in cancer and intellectual disabilities. Results: Inducible RNAi combined with quantitative diGly proteomics was implemented to find HUWE1 substrates. Conclusion: DDIT4 is a substrate of HUWE1. Significance: HUWE1 is a master regulator of cell stress response proteins including DDIT4. Inducible RNAi coupled with diGly proteomics is a valuable strategy for identifying novel E3 ligase substrates.

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Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins

Cited by 49 publications

References 54 publications

A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

Quantitative Lys-ϵ-Gly-Gly (diGly) Proteomics Coupled with Inducible RNAi Reveals Ubiquitin-mediated Proteolysis of DNA Damage-inducible Transcript 4 (DDIT4) by the E3 Ligase HUWE1

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