Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc M.; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T.; Chazin, Walter J.; Kiyokawa, Hiroaki; Yin, Jun

doi:10.1126/sciadv.1701393

Cited by 47 publications

(70 citation statements)

References 73 publications

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“…We expressed human xUB, xUba1, and xUbcH5b or xUbcH7 in HEK293 cells and tested their orthogonality with wt E1 and E2 enzymes. We confirmed that xUB was exclusively loaded on xE1 or xE2 enzymes and it would not cross over to the wt cascade during the transfer (Wang et al, 2017a;Bhuripanyo et al, 2018). These studies established that we could use mutant transplantation to extend the OUT cascade across species and reach different E2 enzymes.…”

Section: The Concept Of Orthogonal Ubiquitin Transfersupporting

confidence: 60%

“…xUbcH5b will not pair with either wt E4B or CHIP to mediate xUB transfer due to the mutations in the N-terminal helix of xUbcH5b that interferes with its binding to the U-box domain of the E3s (Benirschke et al, 2010). To identify U-box mutants complementary with xUbcH5b, we randomized residues in loop1 of E4B U-box that interact with K4E and K8E mutations in xUbcH5b (Bhuripanyo et al, 2018). We used phage selection to identify catalytic active mutants of E4B U-box based on xUB transfer (Fig.…”

Section: The Concept Of Orthogonal Ubiquitin Transfermentioning

confidence: 99%

“…We see potential for transplantation of mutations to expand the OUT cascade within a class of E3s. The E2-binding regions in HECT, U-box, RBR, and RING E3s are homologous Protein Engineering in the Ubiquitin System within each class, and, as proof of principle, we have succeeded in extending the OUT cascade by transplanting mutations from the U-box domain of E4B to CHIP (Bhuripanyo et al, 2018). If transplantation is not efficacious for an E3 of interest, the phage and yeast display methods could be used to engineer individual xE2-xE3 pairs (Wang et al, 2017a;Bhuripanyo et al, 2018).…”

Section: The Concept Of Orthogonal Ubiquitin Transfermentioning

confidence: 99%

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Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond

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Section: The Concept Of Orthogonal Ubiquitin Transfersupporting

confidence: 60%

Section: The Concept Of Orthogonal Ubiquitin Transfermentioning

confidence: 99%

Section: The Concept Of Orthogonal Ubiquitin Transfermentioning

confidence: 99%

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Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond

et al. 2020

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“…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). We expressed the OUT cascade of CHIP in the HEK293 cells with xUB tagged with 69 Histidine and biotin tags (HBT-xUB).…”

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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“…UBE4B has been identified as an E3/E4 ligase that collaborates with Hdm2 to catalyze the polyubiquitination and degradation of the tumor suppressor p53 [32]. UBE4B has also been shown to ubiquitinate additional substrates such as EGFR, Ataxin-3 and OTUB1 [33][34][35]. To date, UBE4B has not been implicated in the ubiquitination of any viral proteins.…”

Section: K63-linked Polyubiquitination and Stabilization And Inhibitimentioning

confidence: 99%

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation

Harhaj

Mohanty²,

Han

et al. 2020

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Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify novel Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax.Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by coimmunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-B target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48-and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-B activation. conjugation factor UBE4B as a novel Tax binding protein that promotes both K48-and K63-linked polyubiquitination of Tax. Knockdown or knockout of UBE4B impairs Taxinduced NF-B activation and triggers apoptosis of HTLV-1 transformed cells. Therefore, UBE4B is an integral host factor that supports HTLV-1 Tax polyubiquitination, NF-B activation and cell survival.

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Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

Abstract: Engineering the ubiquitin transfer cascades by phage display enables an efficient way to profile E3 substrates.

Cited by 47 publications

References 73 publications

Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond

Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond

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

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation

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