Identification of an E3 Ubiquitin Ligase, CUBL, as a Substrate for Protein Arginine Methyltransferase, CARM1, and its Potential Role in Radiation-induced DNA Damage Repair

BRCA1 is a tumor suppressor protein involved in maintaining genomic integrity through multiple functions in DNA damage repair, transcriptional regulation, cell cycle checkpoint, and protein ubiquitination. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase function that plays essential roles in response to DNA damage repair. BRCA1-associated cancers have been shown to confer a hypersensitivity to chemotherapeutic agents. Here, we have studied the functional consequence of the in vitro E3 ubiquitin ligase activity and cisplatin sensitivity of the missense mutation D67Y BRCA1 RING domain. The D67Y BRCA1 RING domain protein exhibited the reduced ubiquitination function, and was more susceptible to the drug than the D67E or wild-type BRCA1 RING domain protein. This evidence emphasized the potential of using the BRCA1 dysfunction as an important determinant of chemotherapy responses in breast cancer.

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Silencing of E3 Ubiquitin Ligase RNF8 Enhances Ionizing Radiation Sensitivity of Medulloblastoma Cells by Promoting the Deubiquitination of PCNA

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504018x15154085345907 ◽

2018 ◽

Vol 26 (9) ◽

pp. 1365-1373

Author(s):

Fei Li ◽

Bin Liu ◽

Xiaolan Zhou ◽

Quan Xu

Keyword(s):

Dna Damage ◽

Ionizing Radiation ◽

Dna Damage Response ◽

Ubiquitin Ligase ◽

Dna Damage Repair ◽

E3 Ubiquitin Ligase ◽

Dna Damage And Repair ◽

Damage Repair ◽

Damage Response ◽

Γ Ray

DNA damage response induced by ionizing radiation (IR) is an important event involved in the sensitivity and efficiency of radiotherapy in human medulloblastoma. RNF8 is an E3 ubiquitin ligase and has key roles in the process of DNA damage and repair. Our study aimed to evaluate the effect of RNF8 in the DNA damage repair induced by IR exposure in medulloblastoma cells. We found that the levels of RNF8 were significantly upregulated by γ-ray irradiation in a dose-dependent manner in medulloblastoma cells and colocalized with γ-H2AX, a sensitive marker of DNA double-strand breaks induced by γ-ray radiation. RNF8 knockdown was observed to enhance the sensitivity of IR in medulloblastoma cells, as evaluated by reduced cell survival. The apoptosis and cell cycle arrest of medulloblastoma cells were dramatically increased by RNF8 suppression after IR treatment. Furthermore, RNF8 inhibition did not affect the protein levels of BRCA1, a crucial protein involved in IR-induced DNA damage repair, but significantly decreased the recruitment of BRCA1 and increased the level of γ-H2AX at DNA damage sites compared to the control. A significant increase in OTM was observed in medulloblastoma cells treated by RNF8 shRNA after exposure to IR, indicating the effect of RNF8 on DNA damage and repair. Additionally, PCNA, a major target for ubiquitin modification during DNA damage response, was found to be monoubiquitinated by E3 ligase RNF8 and might contribute to the low radiosensitivity in medulloblastoma cells. Altogether, our findings may provide RNF8 as a novel target for the improvement of radiotherapy in medulloblastoma.

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Deltex E3 Ubiquitin Ligase 3L confers radioresistance in prostate cancer via Akt pathway

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v19i7.9 ◽

2020 ◽

Vol 19 (7) ◽

pp. 1397-1402

Author(s):

Junyi Xiang ◽

Bodong Lv ◽

Shufeng Fan ◽

Zhitian Zhang ◽

Hui Yang

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Protein Kinase ◽

Cell Survival ◽

Ubiquitin Ligase ◽

Protein Kinase B ◽

Dna Damage Repair ◽

E3 Ubiquitin Ligase ◽

Akt Pathway ◽

Damage Repair

Purpose: To determine the effect of Deltex E3 Ubiquitin Ligase 3L (DTX3L) on the radioresistance of prostate cancer (PCa).Methods: A PCa cell model of radioresistance was established via exposure of cancer cell lines to fractionated radiation. The MTT {(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)} assay and western blotting were performed to evaluate the impact of DTX3L on cell survival and DNA damage repair. The molecular mechanism of action was evaluated by western blotting.Results: DTX3L was elevated in PCa cell lines compared with normal primary prostate epithelial cells (p < 0.01). The survival of PCa cells exposed to radiation was promoted by overexpression of DTX3L, while knockdown of DTX3L abrogated the radioresistance. Moreover, overexpression of DTX3Ldecreased phosphorylation of histone H2AX (γH2AX) and increased Rad51 levels (p < 0.01). However, knockdown of DTX3L reversed the accumulation of γH2AX and Rad51. Phosphorylation of AKT was promoted by DTX3L overexpression, but was reduced by DTX3L knockdown (p < 0.01). Inhibition of AKT (protein kinase B) counteracted the promotion ability of DTX3L on the radioresistance of PCa cells via decreased cell survival ratio, and also inhibited DNA damage repair via accumulation of γ-H2AX and depletion of Rad51 (p < 0.01).Conclusion: DTX3L increases the resistance of prostate cancer to radiotherapy and DNA damage repair in PCa via AKT pathway, indicating a potential therapeutic strategy to overcome radioresistance in PCa. Keywords: DTX3L (Deltex E3 Ubiquitin Ligase 3L), DNA damage, Phosphorylation, Radioresistance, AKT, Protein kinase B, Prostate cancer

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