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  • Pam2CSK4 what Because of the important role of the DDB CUL B

    2019-07-08

    Because of the important role of the DDB1-CUL4B interaction for CUL4B-based E3 ligases, disruption of the DDB1-CUL4B interaction could be an effective approach to treat cancer. Therefore, we developed an in vitro HTS assay based on yeast cell growth inhibition to identify compounds that could disrupt binding between DDB1 and CUL4B. Using a combination of a cell proliferation assay and in vivo ubiquitination analysis in osteosarcoma cells, we validated TSC01131 as an inhibitor of the DDB1-CUL4B complex. Owing to the much weaker cytotoxic activity against hFOB1.19 Pam2CSK4 what than U2OS and MG63 cells, TSC01131 may be a promising candidate for targeting CUL4B-based E3 ligases in the therapy of different cancers, not only in human osteosarcoma. In addition, we also obtained 21 other small molecules that also greatly inhibited yeast cell growth (data not shown). Future efforts will be focused on revealing the underlying mechanisms of these compounds. In summary, this study uncovered three major findings: (1) we identified the individual members of the CRL4BDCAF13 E3 ligase, which could ubiquitinate the tumor suppressor PTEN in vitro and in vivo; (2) we revealed the mechanism of CUL4B overexpression, in which DNA hypermethylation attenuated the transcriptional inhibition of CUL4B by miR-300, leading to its upregulation; and (3) we obtained a small molecule, TSC01131, which could specifically disrupt the DDB1-CUL4B interaction and inhibit osteosarcoma cell proliferation. These results not only provide insight into how CUL4B specifically functions in osteosarcoma cells, but also identify new molecular targets and specific compounds for osteosarcoma therapy.
    Materials and Methods
    Author Contributions
    Acknowledgments This study was supported by two grants from the National Natural Science Foundation of China (81672203 and 81602354) and a grant from the Emerging Frontier Technology Joint Research Program of Shanghai Shen-Kang Hospital Development Center (SHDC12015103).
    Introduction In Escherichia coli, the heat shock response (HSR) [1] is controlled by the major stress sigma factor σ32[2], which guides RNA polymerase to HSP gene promoters under heat stress [3], [4], [5]. During the HSR induction phase, σ32 is primarily regulated at the translational level and σ32 activity and stability increase [6]. During the adaptation phase of the HSR, the cytoplasmic DnaK machine and GroEL/GroES negatively regulate σ32 activity thus turning off the HSR [7], [8], [9]. During this stage, σ32 stability is primarily controlled by the membrane-anchored FtsH protease [10], [11]. Recent studies have demonstrated that the interaction between the Signal Recognition Particle (SRP) and σ32 is indispensable for σ32 localization in the cell membrane [12], [13]. In addition to endogenous HSPs, the heterologous expression of eukaryotic E3 ligases increases E. coli cell viability at high temperatures. For example, Brassica napus E3 ligase, BnTR1, and Arabidopsis thaliana E3 ligases, AT1G02860 and AT2G02960, can interact with σ32 and DnaK to maintain the level of σ32 in the cell. Previous research suggested that endogenous σ32 could be covalently modified by the ubiquitin-like protein ThiS in vivo[14] and K. Heran Darwin [15] found that the prokaryotic ubiquitin-like protein (Pup) of Mycobacterium tuberculosis could pupylate at least 51 E. coli proteins. Thus, it is possible that these E3 ligases from plants could ubiquitinate σ32 or DnaK. This work shows that BnTR1, AT2G02960, and AT1G02860 have E3 ligase activity. Using an in vitro ubiquitination assay, BnTR1 and AT1G02860 were shown to ubiquitinate σ32 instead of DnaK; however, AT2G02960 did not have a similar function. To verify the functional motif of the E3 ligases, a RING finger domain mutation was constructed for BnTR1, AT2G02960, and AT1G02860. Immunoblot analysis indicated that the RING finger domain was essential for the function of E3 ligase activity. These results imply that the plant ubiquitylation system likely evolveds from prokaryotes.