• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • MRS 2179 tetrasodium salt br Results br Discussion Cullin


    Discussion Cullin-based E3 ligases have been shown to play critical roles in tumorigenesis and to contribute to diverse biological processes in human cancers. CUL4A and CUL4B, two paralogs in the human genome, share high sequence similarity. The CUL4A-based E3 ligases have been reported to ubiquitinate a number of different substrates, including DDB2, p12, CDT1, p21, and p27.21, 22, 23, 24, 25 However, to date, the role of CUL4B in cancer, the substrates of CRL4B E3 ligases, and the underlying mechanism of CUL4B overexpression are far from being fully elucidated. In this study, we discovered that CUL4B formed an E3 ligase complex with DDB1, RBX1 and DCAF13, and the CRL4BDCAF13 E3 ligase recognized PTEN, a tumor suppressor, as a substrate for ubiquitination. Moreover, we also found that miR-300, an miRNA that directly targets the 3′ UTR of CUL4B, was significantly downregulated via a DNA hypermethylation mechanism in its promoter region in human osteosarcoma cells. The downregulation of miR-300 attenuated its transcriptional inhibition of CUL4B, thereby resulting in the overexpression of CUL4B. Either ectopic expression of miR-300 or treatment with the DNA methylation inhibitor AZA was capable of reducing PTEN ubiquitination, eventually resulting in the accumulation of PTEN. Importantly, we also identified a small molecule, TSC01131, which could directly abolish the CUL4B-DDB1 interaction, leading to reduced stability of CRL4BDCAF13 E3 ligase. Taken together, our results identified a new E3 ligase that recognized PTEN as a specific substrate in human osteosarcoma cells, and we also revealed the underlying mechanism for CUL4B overexpression and identified a small molecule that specifically targets the CRL4BDCAF13 E3 ligase (Figure 7). These results explain the mechanism of CUL4B in osteosarcoma and provide potential new therapeutic targets for future osteosarcoma treatment. Although the amino MRS 2179 tetrasodium salt sequences of CUL4A and CUL4B share 82% identity, the existing studies have shown that these two proteins do not have significant functional redundancy. Elevated expression of CUL4A has been observed in a variety of cancer cells, such as breast cancer, ovarian cancer, liver cancer, and medulloblastoma. CRL4A-based E3 ubiquitin ligase can recognize a series of substrate proteins, such as DDB2, XPC, UNG2, and SMUG1, as well as histones H2A, H3, and H4, which are widely involved in DNA damage and repair process. In addition, CRL4ACTD2 E3 ligase can recognize a number of cell-cycle regulatory proteins, such as CTD1, p21, and Chk1, as substrates to regulate cancer cell-cycle progression. Interestingly, CRL4A-based E3 ligases are also involved in the p53 and Wnt signaling pathways. However, the function of CUL4B in cancer remains unclear. Previous studies mainly focused on its function in cell development; for instance, CUL4B mutations lead to defects in nervous system and cardiac development.47, 48 In recent years, studies have also shown that CUL4B plays an important role in the process of tumorigenesis, and high expression of CUL4B has been reported in many cancers. For example, in esophageal cancer cells, CRL4B-based E3 ubiquitin ligase can ubiquitinate histone H2A and can coordinate with multi-comb inhibitory complex PRC2 to promote tumor cell proliferation and invasion. Silencing CUL4B in HeLa cells can lead to cyclin E aggregation and can cause cell-cycle arrest in S phase, thereby inhibiting cell proliferation. In prognosis studies of colorectal cancer patients, high expression of CUL4B was associated with a poor prognosis, but the molecular mechanism was unclear. Moreover, our previous results also showed that CUL4B is upregulated in human osteosarcoma cells, and its silencing could effectively inhibit osteosarcoma cell proliferation and induce apoptosis. In the current investigation, we identified the individual components of CRL4BDCAF13 E3 ligase through Y2H screening and LC-MS/MS methods, and we also identified the specific target of this E3 ligase: PTEN. By carefully analyzing the ubiquitination pattern of PTEN, we found that PTEN is modified by polyubiquitination through our in vitro and in vivo studies, indicating that more than one lysine (K) site is modified. Through amino acid sequence analysis, we found that PTEN contains a total of 33 K sites; however, these K sites are distributed everywhere, making it difficult to find the modified sites in our case. Some studies have noted that the C terminus of PTEN plays a crucial role in antagonizing NEDD4-1-mediated ubiquitination, and some K sites, such as K13 and K289, were reportedly modified in 293 cells. However, we cannot conclude which sites were modified in our system. We are currently mapping the modified sites through point mutagenesis.