image: (A) UBR5 was positively correlated with Snail in colon adenocarcinoma and rectal adenocarcinoma. Correlation analysis for TCGA and GTEx on the GEPIA website showed a correlation coefficient of R = 0.26, P = 4.4e-13. P < 0.01 denoted statistical significance. (B) The expression levels of UBR5 and Snail correlated with colorectal cancer (CRC) stages. The UBR5 and Snail mRNA levels based on pathological stages were analyzed using the GEPIA2 violin-plots in colorectal tumors. (C) Co-immunoprecipitation assay showed that UBR5 interacted with Snail. HEK293T cells were transfected with Myc-tagged UBR5 and Flag-tagged Snail and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (D) Co-localization of UBR5 and Snail in the nucleus. Immunofluorescence assay probe co-localization of UBR5 (red) and Snail (green). Scale bar: 50 μm. (E) UBR5 interacted with Snail through the HECT domain. A schematic of various UBR5 truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various UBR5 truncations. (F) Snail interacted with UBR5 through the zinc-figure domain. A schematic of various Snail truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various Snail truncations. (G) Molecular docking of Snail zinc-figure domain (amino acids 151–264) and UBR5 HECT domain (amino acids 2453–2799) truncation protein. (H) The HECT domain of UBR5 interacted with Snail in the co-immunoprecipitation assay. HEK293T cells were transfected with wild-type and truncated Myc-tagged UBR5, as well as Flag-tagged Snail, and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail or anti-UBR5 antibodies.
Credit: Xinyue Zhao, Ruiying Liu, Zhihui Han, Zehao Li, Ling Mei, Yuyang Liu, Xueqi Fu, Yue Jin
In a recent study published in Genes & Diseases, researchers from Jilin University identify UBR5, a HECT-domain containing E3 ubiquitin ligase, as a critical tumor suppressor that governs metastatic potential by modulating the stability of Snail, a master transcriptional regulator of EMT.
Using affinity purification-mass spectrometry, the authors first identified UBR5 as one of the Snail-interacting proteins. Clinical and transcriptomic analyses using TCGA and GTEx datasets revealed a significant stage-specific expression pattern in CRC; specifically, in stage II CRC, UBR5 expression was notably diminished while Snail levels were elevated, suggesting that the loss of UBR5 facilitates Snail stabilization during early metastatic events.
The study also established that the UBR5-Snail interaction is highly specific; while UBR5 effectively binds to Snail, it does not interact with the closely related family member Slug (Snail2). Domain mapping and molecular docking further elucidate that the HECT domain (amino acids 2453–2799) of UBR5 serves as the critical binding site for the C-terminal zinc finger domain of Snail. These findings distinguish UBR5 from previously identified RING-family E3 ligases and characterize it as a distinct post-translational regulator of the Snail-mediated metastatic cascade in CRC.
The authors demonstrated that UBR5 maintains the epithelial phenotype by targeting Snail for K48-linked polyubiquitination and proteasomal degradation – a mechanism strictly contingent upon GSK-3β-mediated Snail phosphorylation and the structural integrity of the UBR5 HECT domain, which directly interacts with the Snail C-terminal zinc finger domain.
The loss of UBR5 triggers a molecular “switch” characterized by Snail stabilization, E-cadherin downregulation, and increased mesenchymal marker expression, collectively enhancing cellular motility and invasiveness. Furthermore, in vivo xenograft models confirmed that UBR5 deficiency not only accelerates tumor growth but also promotes local tissue infiltration, establishing the UBR5-Snail axis as a critical post-translational checkpoint for suppressing metastasis.
The Cys2768 residue within the UBR5 HECT domain is essential for regulating Snail-mediated metastasis. Cycloheximide pulse-chase and ubiquitination assays revealed that the UBR5 C2768S catalytic mutation completely abolishes the physical interaction between UBR5 and Snail, eliminating the ligase’s ability to accelerate Snail protein turnover.
This was corroborated in vivo, where tumors overexpressing wild-type UBR5 exhibited significant regression and clear encapsulation, while C2768S-mutant groups showed aggressive infiltration into adjacent muscle tissues.
Clinical datasets from TCGA and GTEx further confirm that UBR5 is significantly downregulated in CRC tissues and that its expression serves as a favorable prognostic indicator for relapse-free survival.
The study concludes that the UBR5-Snail axis serves as a critical post-translational checkpoint, where the catalytic integrity of UBR5 at site 2768 is essential for maintaining the epithelial state, suppressing the metastatic cascade and improving relapse-free survival in patients with CRC.
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