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A Comprehensive Method for Detecting Ubiquitinated Substrates Using TR-TUBE

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A Comprehensive Method for Detecting Ubiquitinated Substrates Using TR-TUBE A comprehensive method for detecting ubiquitinated substrates using TR-TUBE Yukiko Yoshidaa,1, Yasushi Saekib, Arisa Murakamia,b, Junko Kawawakia, Hikaru Tsuchiyab, Hidehito Yoshiharab, Mayumi Shindoc, and Keiji Tanakab,1 aProtein Metabolism Project, bLaboratory of Protein Metabolism, and cCenter for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan Edited by Aaron Ciechanover, Technion-Israel Institute of Technology, Bat Galim, Haifa, Israel, and approved March 10, 2015 (received for review November 21, 2014) The identification of substrates for ubiquitin ligases has remained of ubiquitinated proteins, has been developed for global pro- challenging, because most substrates are either immediately de- teomic applications aimed at identifying ubiquitinated substrates graded by the proteasome or processed by deubiquitinating (8, 9). Although a few quantitative proteomics studies have enzymes (DUBs) to remove polyubiquitin. Although a methodol- identified a particular ubiquitin ligase substrate using stable ogy that enables detection of ubiquitinated proteins using ubiq- isotope labeling utilizing amino acids in cell culture and the anti- uitin Lys-e-Gly-Gly (diGly) remnant antibodies and MS has been diGly antibody (10), these examples required large quantities of developed, it is still insufficient for identification and characteriza- samples and advanced techniques. tion of the ubiquitin-modified proteome in cells overexpressing Tandem ubiquitin-binding entity(ies) (TUBE) based on ubiquitin- a particular ubiquitin ligase. Here, we show that exogenously associated domains have been developed for isolation of poly- expressed trypsin-resistant tandem ubiquitin-binding entity(ies) ubiquitinated proteins from cell extracts (11). Notably, TUBE (TR-TUBE) protect polyubiquitin chains on substrates from reagents protect polyubiquitin-conjugated proteins in cell lysates DUBs and circumvent proteasome-mediated degradation in cells. from both proteasomal degradation and deubiquitinating en- TR-TUBE effectively associated with substrates ubiquitinated by an zymes (DUBs) as efficiently as specific inhibitors of these en- exogenously overexpressed ubiquitin ligase, allowing detection zymes (11). In this paper, we applied the TUBE technology to in of the specific activity of the ubiquitin ligase and isolation of its vivo capture of ubiquitinated proteins. To develop a versatile substrates. Although the diGly antibody enabled effective identi- method for identifying substrates of a specific ubiquitin ligase, we fication of ubiquitinated proteins in cells, overexpression of an designed a mammalian expression vector encoding a FLAG- ubiquitin ligase and treatment with a proteasome inhibitor did tagged trypsin-resistant (TR) TUBE, which protects ubiquitin not increase the level of diGly peptides specific for the ligase rel- chains from trypsin digestion under native conditions. Using two ative to the background level of diGly peptides, probably due to enrichment methods, TR-TUBE and the anti-diGly antibody, we deubiquitination. By contrast, in TR-TUBE–expressing cells, the succeeded in identifying the target substrates of the uncharac- level of substrate-derived diGly peptides produced by the over- terized F-box protein FBXO21. expressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin li- Results gases using two enrichment strategies, TR-TUBE and diGly remnant Protection of Polyubiquitin Chains on Substrates by TR-TUBE. Our antibodies, coupled with MS. Using this method, we identified tar- method is based on stabilization of ubiquitinated substrates in get substrates of FBXO21, an uncharacterized F-box protein. vivo by masking of ubiquitin chains with exogenously expressed ubiquitin-binding protein | ubiquitin ligase | ubiquitination Significance osttranslational modification by ubiquitin regulates diverse The identification of specific ubiquitin ligase–substrate pairs is Pprocesses in cells (1, 2). Ubiquitination is catalyzed by three crucial for understanding the roles of protein ubiquitination in types of enzymes—E1, E2, and E3, with the selectivity for the the regulation of diverse biological processes. Despite the target protein provided by E3 ubiquitin ligases. Although the development of various methodologies for substrate identi- human genome encodes more than 600 ubiquitin ligases, many fication, it remains challenging to determine ubiquitin ligase of them remain to be studied (3). The Skp1–Cul1–F-box protein substrates. Based on previously described tandem ubiquitin- (SCF) complex, one of the best-characterized ubiquitin ligases, is binding entity(ies) (TUBE), we designed the trypsin-resistant composed of three invariable components (Skp1, Cul1, and (TR)-TUBE for expression in cells. The coexpression of TR-TUBE Rbx1) and a variable component F-box protein that serves as the with an ubiquitin ligase stabilizes the ubiquitinated substrates substrate recognition module. Among the over 70 F-box proteins by masking the ubiquitin chains. Using a combination of two found in humans, less than half have been characterized (4). strategies for enriching ubiquitinated substrates, TR-TUBE and The identification of substrates for a specific ubiquitin ligase anti–Lys-e-Gly-Gly antibody, we successfully identified specific has been challenging despite considerable efforts. To date, the ubiquitin ligase–substrate pairs. Our methodology provides an physical interaction between an ubiquitin ligase and its substrates effective means for the identification of ubiquitin ligase sub- has been exploited as the major approach for substrate identi- strates and the detection of ubiquitin ligase activity. fication (5–7). In these studies, immunoprecipitation followed by MS has been used to isolate ligase–substrate complexes. How- Author contributions: Y.Y., Y.S., and K.T. designed research; Y.Y., Y.S., A.M., J.K., H.T., and H.Y. performed research; Y.Y., Y.S., H.T., H.Y., and M.S. analyzed data; and Y.Y., Y.S., and ever, there are several difficulties associated with this approach: K.T. wrote the paper. – Most ligase substrate interactions are generally too weak and The authors declare no conflict of interest. transient to isolate the substrates by immunoprecipitation, and This article is a PNAS Direct Submission. the abundances of relevant in vivo substrates are often low due 1To whom correspondence may be addressed. Email: [email protected] or to proteasomal degradation. [email protected]. Recently, an antibody that recognizes the ubiquitin remnant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. motif Lys-e-Gly-Gly (diGly), which is exposed upon tryptic digestion 1073/pnas.1422313112/-/DCSupplemental. 4630–4635 | PNAS | April 14, 2015 | vol. 112 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1422313112 Downloaded by guest on September 24, 2021 WCL B posttransfection (h) 0 24 4872 48 C 100 100 A TR-TUBE + + + + emp 80 TR-TUBE 80 TR-TUBE mutant + + + 60 mutant 60 MG132 (h) -------- 48 40 40 kDa 20 20 Viability (%) DUB 0 Viability (%) 0 E3 IP: TR-TUBE 188 Ubiquitin 0 24 48 72 0 4 8 98 posttransfection (h) MG132 treatment (h) Ub Lyse 62 conjugates Ub Ub 49 Ub Proteasome Ub 38 Ub TR-TUBEUb Ub Ub 28 Substrate Ub TR-TUBEUb 17 Substrate Ub 14 TR-TUBE Substrate E IP: αFLAG 6 Ub FLAG-tagged Ub TR-TUBE α α D WCL IP: FLAG WCL IP: FLAG αUbiquitin HA-tagged emp p27 emp p27 FLAG-TR-TUBE FLAG-TR-TUBE HA-Skp2 - + - + - + - + posttransfection (h)24 48 72 24 48 72 24 48 72 24 48 72 MG132 -+-+-+-+ -+-+-+-+ kDa HA-Skp2 -+-+-+ -+-+- + - + - + - + - + - +-+ 250 kDa kDa 150 188 (Ub)n 98 250 100 (Ub)n 75 62 150 -p27 49 100 50 38 75 -p27 28 37 17 50 37 25 14 20 6 25 20 12 34 4 5 6 7 8 9 10 11 1231 1 15 16 αUbiquitin αp27 αp27 Fig. 1. Protection of polyubiquitin chains on substrates by TR-TUBE. (A) TR-TUBE method for isolation of ubiquitinated substrates. Polyubiquitin chains on substrates are masked by exogenously expressed TR-TUBE, and thereby protected from DUBs and the proteasome. Ubiquitinated proteins are enriched by immunoprecipitation (IP) of TR-TUBE from cells expressing E3 ubiquitin ligase and TR-TUBE. Exogenously expressed proteins are shown in red. Ub, ubiquitin. (B) Accumulation of ubiquitin conjugates in TR-TUBE–expressing cells. 293T cells were transfected with FLAG-TR-TUBE or ubiquitin-binding–deficient FLAG- TR-TUBE mutant plasmid, and the transfected cells were harvested at the indicated times. Cells transfected with HA-empty (emp) vector were treated with 10 μM MG132 for the indicated time before harvesting. Whole-cell lysates (WCLs) were analyzed by immunoblotting using antiubiquitin antibody. (C) Effect of TR-TUBE expression or MG132 treatment on cell viability, as determined by propidium iodide staining. Three independent plates of transfected or MG132 cells were analyzed. Error bars represent means ± SEM. (D) Detection of ubiquitin conjugates and ubiquitinated endogenous p27. Cells (1.3 × 106) were cotransfected with 3.5 μg of FLAG-TR-TUBE and 3.5 μg of HA-empty or HA-Skp2 expression plasmids, and the transfected cells were harvested at the indicated times. WCLs and anti-FLAG immunoprecipitates were analyzed by immunoblotting. The arrow indicates the position of p27. (E) Detection of ubiquitination of endogenous and overexpressed p27. Cells expressing FLAG-ubiquitin or FLAG-TR-TUBE with or without HA-Skp2 and/or HA-p27 were treated with or without MG132, and
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