Abstract
BackgroundTargeting ubiquitin-dependent proteolysis is one of the strategies in cancer therapy. CRLCDT2 and CRLDDB2 are two key E3 ubiquitin ligases involved in DNA replication and DNA damage repair. But CDT2 and DDB2 are opposite prognostic factors in kinds of cancers, and the underlining mechanism needs to be elucidated.MethodsSmall interfering RNAs were used to determine the function of target genes. Co-immunoprecipitation (Co-IP) was performed to detect the interaction between DDB2 and CDT2. Immunofluorescence assays and fluorescence activating cell sorting (FACS) were used to measure the change of DNA content. In vivo ubiquitination assay was carried out to clarify the ubiquitination of CDT2 mediated by DDB2. Cell synchronization was performed to arrest cells at G1/S and S phase. The mechanism involved in DDB2-mediated CDT2 degradation was investigated by constructing plasmids with mutant variants and measured by Western blot. Immunohistochemistry was performed to determine the relationship between DDB2 and CDT2. Paired two-side Student’s t-test was used to measure the significance of the difference between control group and experimental group.ResultsKnockdown of DDB2 stabilized CDT2, while over-expression of DDB2 enhanced ubiquitination of CDT2, and subsequentially degradation of CDT2. Although both DDB2 and CDT2 contain PIP (PCNA-interacting protein) box, PIP box is dispensable for DDB2-mediated CDT2 degradation. Knockdown of PCNA had negligible effects on the stability of CDT2, but promoted accumulation of CDT1, p21 and SET8. Silencing of DDB2 arrested cell cycle in G1 phase, destabilized CDT1 and reduced the chromatin loading of MCMs, thereby blocked the formation of polyploidy induced by ablation of CDT2. In breast cancer and ovarian teratoma tissues, high level of DDB2 was along with lower level of CDT2.ConclusionsWe found that CRL4DDB2 is the novel E3 ubiquitin ligases of CDT2, and DDB2 regulates DNA replication through indirectly regulates CDT1 protein stability by degrading CDT2 and promotes the assembly of pre-replication complex. Our results broaden the horizon for understanding the opposite function of CDT2 and DDB2 in tumorigenesis, and may provide clues for drug discovery in cancer therapy.
Highlights
Targeting ubiquitin-dependent proteolysis is one of the strategies in cancer therapy
DNA damage binding protein 2 (DDB2) mediated degradation of CDT2 have different effect on CDT1, p21 and SET8 Since depletion of DDB2 can rescue DNA re-replication induced by CDT2 knockdown, we examined the changes of protein levels of CDT1, p21, and SET8—three canonical substrates of CRL4CDT2 ubiquitin ligase [5, 8, 43]— after DDB2 silencing, to figure out the mechanism that DDB2 affects DNA re-replication
We found that deletion of DDB2 significantly reduced the protein level of CDT1, and p21was slightly decreased while SET8 was not affected (Fig. 4a), which indicated that degradation of CDT2 mediated by DDB2 has main effect on CDT1 but not p21 and SET8
Summary
Targeting ubiquitin-dependent proteolysis is one of the strategies in cancer therapy. C RLCDT2 and CRLDDB2 are two key E3 ubiquitin ligases involved in DNA replication and DNA damage repair. CRL4CDT2 ubiquitin ligase regulates cell proliferation by degrading important substrates such as CDT1, p21 and SET8, which involves in DNA replication licensing, cell cycle regulation, and chromatin modification [5,6,7]. Silencing of CDT2 induces the apoptotic death of human cancer cells from different tissues, but not non-transformed human cells and primary cells, which may due to the replicative stress and DNA damage [13]. MLN4924 can inhibit the activity of CRL4CDT2 ubiquitin ligase to stabilize CDT1 and trigger checkpoint activation, apoptosis, and senescence in cancer cells [15]. Knockdown of CDT2 but not DCAF1 phenocopies the effects of MLN4924, and silencing of CDT1 partially rescues apoptotic death induced by MLN4924 [16], while the pharmacological effect of MLN4924 is independent of p21 or SET8 [14], indicating special role of CDT2 and CDT1 in cancer development
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