Background
Ribosomal L1 domain-containing protein 1 (RSL1D1) is a nucleolar protein that is essential in cell proliferation. In the current opinion, RSL1D1 translocates to the nucleoplasm under nucleolar stress and inhibits the E3 ligase activity of HDM2 via direct interaction, thereby leading to stabilization of p53.
Methods
Gene knockdown was achieved in HCT116p53+/+, HCT116p53−/−, and HCT-8 human colorectal cancer (CRC) cells by siRNA transfection. A lentiviral expression system was used to establish cell strains overexpressing genes of interest. The mRNA and protein levels in cells were evaluated by qRT-PCR and western blot analyses. Cell proliferation, cell cycle, and cell apoptosis were determined by MTT, PI staining, and Annexin V-FITC/PI double staining assays, respectively. The level of ubiquitinated p53 protein was assessed by IP. The protein-RNA interaction was investigated by RIP. The subcellular localization of proteins of interest was determined by IFA. Protein-protein interaction was investigated by GST-pulldown, BiFC, and co-IP assays. The therapeutic efficacy of RSL1D1 silencing on tumor growth was evaluated in HCT116 tumor-bearing nude mice.
Results
RSL1D1 distributed throughout the nucleus in human CRC cells. Silencing of RSL1D1 gene induced cell cycle arrest at G1/S and cell apoptosis in a p53-dependent manner. RSL1D1 directly interacted with and recruited p53 to HDM2 to form a ternary RSL1D1/HDM2/p53 protein complex and thereby enhanced p53 ubiquitination and degradation, leading to a decrease in the protein level of p53. Destruction of the ternary complex increased the level of p53 protein. RSL1D1 also indirectly decreased the protein level of p53 by stabilizing HDM2 mRNA. Consequently, the negative regulation of p53 by RSL1D1 facilitated cell proliferation and survival and downregulation of RSL1D1 remarkably inhibited the growth of HCT116p53+/+ tumors in a nude mouse model.
Conclusion
We report, for the first time, that RSL1D1 is a novel negative regulator of p53 in human CRC cells and more importantly, a potential molecular target for anticancer drug development.
Kaempferol, an important flavonol, has numerous health-beneficial bioactivities and possesses a great potential for application in medicine, food, and cosmetics industries. To improve the production of kaempferol in an in vitro synthetic biosystem, we designed and constructed a panel of bifunctional enzymes by fusing the flavanone 3-hydroxylase (AtF3H) and the flavonol synthase (AtFLS1) of Arabidopsis thaliana with different orientation and different peptide linker type and length. By comparing the output of kaempferol, we obtained a highly active bifunctional enzyme AtF3H-(GGGGS) 2 -AtFLS1 with a Km value of 0.129 ± 0.016 mM. After optimization of a series of reaction parameters, kaempferol was produced at 100.54 ± 0.54 mg/L, the currently highest kaempferol output in an in vitro synthetic biosystem, and the substrate conversion rate was 68.26% ± 0.05%. In addition, we observed substrate inhibition for the AtFLS1, which eventually limited the production of kaempferol. This study provides a highly active biocatalyst for production of kaempferol and an insight into biosynthesis of other valuable molecules.
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