Orthogonal Ubiquitin Transfer through Engineered E1-E2 Cascades for Protein Ubiquitination

Zhao, Bo; Bhuripanyo, Karan; Zhang, Keya; Kiyokawa, Hiroaki; Schindelin, Hermann; Yin, Jun

doi:10.1016/j.chembiol.2012.07.023

Cited by 31 publications

(50 citation statements)

References 37 publications

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“…The xE1-xE2-xE3 cascade would exclusively transfer an affinity tagged UB mutant (xUB) to xE3 and then to its substrate proteins in the cell. We previously engineered xUB-xUba1 and xUba1-xUbcH5b pairs to enable the exclusive transfer of xUB to xUbcH5b (Zhao et al, 2012). We recently used phage display to engineer an xUbcH5b-xCHIP pair that would deliver xUB to CHIP and then to its substrate proteins (Bhuripanyo et al, 2018).…”

Section: Mot-2 Is a Substrate For The Chip E3 Ubiquitin Ligasementioning

confidence: 99%

UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

et al. 2018

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Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug‐induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin‐2 (mot‐2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot‐2 enhances tumor cell invasion and migration. Thus, mot‐2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin‐like protein called UBXN2A in the regulation of mot‐2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull‐down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70‐interacting protein (CHIP)‐dependent ubiquitination of mot‐2. We subsequently showed that UBXN2A increases proteasomal degradation of mot‐2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot‐2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot‐2 in cancer cells. Consistent with the in vitro results, UBXN2A+/− mice exhibited selective elevation of mot‐2 in colon tissues. An in vitro Anti‐K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot‐2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A‐mot‐2 complex in tumors in an azoxymethane/dextran sulfate sodium‐induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot‐2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot‐2‐enriched tumors. This finding validates the UBXN2A‐CHIP axis as a novel and potential therapeutic target in CRC.

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Section: Mot-2 Is a Substrate For The Chip E3 Ubiquitin Ligasementioning

confidence: 99%

UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

et al. 2018

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“…We profiled the C-terminal sequences of UB that are catalytically active with NAE based on a phage display method we developed to engineer UB recognition by UAE (Figure 1b) [24]. In this method we displayed a UB library with randomized C-terminal sequences on the surface of the M13 phage.…”

Section: Resultsmentioning

confidence: 99%

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

et al. 2013

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The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a “gate-keeping” role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.

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“…Because biotinylated modifiers are provided as in vitro generated E2~modifier conjugates (Fig. 2), there is no need for time-consuming genetic or protein engineering of the source material, which is required for biotinylation approaches in living cells 46,71 or for engineered E1-E2-E3 cascades 30 . Since most E1 and E2 enzymes as well as ubiquitin and UBLs are commercially available or can be easily expressed in E. coli, performing E2~dID does not require extensive biochemical expertise.…”

Section: Discussionmentioning

confidence: 99%

“…However, the dynamic and reversible nature of these modifications, the weak and/or transient interaction between ligase and substrate, the significant degree of redundancy and multiplicity between E1, E2 and E3 enzymes, and the rapid destruction of many ubiquitylated proteins still present significant technical challenges in identifying E3 ligase substrates 20 . Current approaches to define enzyme-substrate relations include yeast two-hybrid 21,22 , protein microarrays 23,24 , substrate trapping [25][26][27][28] , biotin-dependent proximity labelling (BioID) 29 , and engineered ubiquitin enzyme cascades 30 . Alternatively, the abundance of modified substrates can be increased by overexpressing the E3 ligase of interest 31,32 .…”

Section: Families Initially Form Anmentioning

confidence: 99%

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

Bakos

Gak

et al. 2018

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Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models. E2~dID with APC/CAs a proof of principle, we set up E2~dID in HeLa cells and aimed to identify established mitotic substrates of APC/C. APC/C is essential for chromosome segregation in eukaryotic cells by targeting CCNB1 and PTTG1 for proteasomal degradation. During mitosis and G1 phase, the E2 enzymes UBE2C and UBE2S are required for initiating and elongating mainly lysine 11-linked ubiquitin chains on APC/C substrates, respectively 37-39 . First, we generated bioUBB by in vitro biotinylation of ubiquitin on a small N-terminal linked AVI-tag 40 (Supplementary Figure 1a). Alternatively, bioUBB can be obtained from commercial sources. To generate E2~bioUBB thioesters that support APC/Cdependent ubiquitination, we combined recombinant human E1 (UBA1), E2 (UBE2C), bioUBB and ATP in in vitro charging reactions. While UBE2C~bioUBB thioesters were readily formed, UBE2C became auto-ubiquintinated during charging as indicated by a mobility shift of UBE2C on SDS-PAGE

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Orthogonal Ubiquitin Transfer through Engineered E1-E2 Cascades for Protein Ubiquitination

Cited by 31 publications

References 37 publications

UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

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