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Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.

ABSTRACT: Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.

SUBMITTER: Shin YJ

PROVIDER: S-EPMC4571616 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.

Shin Yeong Ju YJ Park Seung Kyun SK Jung Yoo Jung YJ Kim Ye Na YN Kim Ki Sung KS Park Ok Kyu OK Kwon Seung-Hae SH Jeon Sung Ho SH Trinh Le A le A Fraser Scott E SE Kee Yun Y Hwang Byung Joon BJ

Scientific reports 20150916

Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtua ...[more]

PMID: 26373678

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Project description:BackgroundRecurrence and chemoresistance constitute the leading cause of death in colorectal cancer (CRC). Thus, it is of great significance to clarify the underlying mechanisms and identify predictors for tailoring adjuvant chemotherapy to improve the outcome of CRC.MethodsBy screening differentially expressed genes (DEGs), constructing random forest classification and ranking the importance of DEGs, we identified membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) as an important gene in CRC recurrence. Immunohistochemical and western blot assays were employed to further detect MAGI3 expression in CRC tissues and cell lines. Cell counting kit-8, plate colony formation, flow cytometry, sub-cutaneous injection and azoxymethane plus dextran sulfate sodium induced mice CRC assays were employed to explore the effects of MAGI3 on proliferation, growth, cell cycle, apoptosis, xenograft formation and chemotherapy resistance of CRC. The underlying molecular mechanisms were further investigated through gene set enrichment analysis, quantitative real-time PCR, western blot, co-immunoprecipitation, ubiquitination, GST fusion protein pull-down and immunohistochemical staining assays.ResultsOur results showed that dysregulated low level of MAGI3 was correlated with recurrence and poor prognosis of CRC. MAGI3 was identified as a novel substrate-binding subunit of SKP1-Cullin E3 ligase to recognize c-Myc, and process c-Myc ubiquitination and degradation. Expression of MAGI3 in CRC cells inhibited cell growth, promoted apoptosis and chemosensitivity to fluoropyrimidine-based chemotherapy by suppressing activation of c-Myc in vitro and in vivo. In clinic, the stage II/III CRC patients with MAGI3-high had a significantly good recurrence-free survival (~ 80%, 5-year), and were not necessary for further adjuvant chemotherapy. The patients with MAGI3-medium had a robustly good response rate or recurrence-free survival with fluoropyrimidine-based chemotherapy, and were recommended to undergo fluoropyrimidine-based adjuvant chemotherapy.ConclusionsMAGI3 is a novel E3 ubiquitin ligase by degradation of c-Myc to regulate CRC development and may act as a potential predictor of adjuvant chemotherapy for CRC patients.

Dataset Information

Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.

Publications

Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.

Similar Datasets

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