Mutations in DNA binding domain of p53 impede RSL1D1-p53 interaction to escape from degradation in human colorectal cancer cells.

Different from the nucleolus-specific localization in some types of cancer cells, ribosomal L1 domain-containing protein 1 (RSL1D1) distributes throughout the nucleus in human colorectal cancer (CRC) cells. RSL1D1 directly interacts with DNA binding domain (aa 93-292) of wild-type p53 (p53-WT) and thereby recruits p53 to HDM2. The ensuing formation of RSL1D1/HDM2/p53 complex enhances p53 ubiquitination and decreases the protein level of p53 in CRC cells. In this study, we investigated the interaction between RSL1D1 and mutant p53 proteins. We first corroborated that aa 93-224 of p53 is a more precise domain for RSL1D1 binding and mutation in either aa 93-224 or aa 225-292 domain of p53 affects RSL1D1-p53 interaction. R175H mutated p53 does not interact with RSL1D1, whereas R273H mutated p53 still can bind to RSL1D1 but showing a remarkably decreased affinity than p53-WT. Although p53-R273H retains a weakened binding affinity with RSL1D1, it can hardly be recruited to HDM2 by RSL1D1 in HCT116 CRC cells. Accordingly, RSL1D1 loses its capacity to negatively regulate either R175H or R273H p53 mutant via directly interaction in HCT116 cells, thereby facilitating p53 mutants to accumulate and gain oncogenic function. Our findings help explain why mutant p53 proteins are more stable than p53-WT in CRC cells.

Programmed cell death 11 modulates but not entirely relies on p53-HDM2 loop to facilitate G2/M transition in colorectal cancer cells.

We previously described a nucleolar protein RSL1D1 but distributed throughout the nucleus in HCT116 colorectal cancer (CRC) cells to facilitate G1/S transition by inhibiting p53 signaling. Here, we found another nucleolar protein, programmed cell death 11 (PDCD11), also with an "Extra-nucleolar" localization in CRC cells but to regulate G2/M checkpoint. This protein directly interacts with p53 and HDM2 in the nucleoplasm, thereby recruiting p53 to HDM2 for ubiquitination and degradation. The ensuing downregulation of p53 increases the CDK1 level to help the cells pass G2/M checkpoint. Upon DNA damage stress, PDCD11 gains the power to upregulate CDK1 independently of p53. Beyond these, PDCD11 also upregulates CDC25C in a p53-independent manner to dephosphorylate CDK1 to facilitate G2/M transition. Downregulation of PDCD11 greatly reduced cancer cell growth in vitro and in vivo, additionally sensitized cells to DNA damage signals, highlighting that PDCD11 is a crucial driving factor of CRC and a potential target for cancer treatment.

Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells.

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.

Theoretical analysis and enhanced nitrogen removal performance of step-feed SBR.

To achieve high nitrification and denitrification rates, step-feed SBR with multiple aerobic and anoxic phases was adopted to study nitrogen removal performance. Theoretical analysis of step-feed SBR was presented first, from which feeding steps and C/N ratio had significant influences on nitrogen removal performance. Total nitrogen removal efficiency would increase with increasing of feeding steps, while the increasing extent was not distinct with feeding steps above 4. At a given feeding step, nitrogen removal efficiency would also increase with increasing of C/N ratio. Experimental work was conducted in a lab-scale SBR to investigate practical effects of these critical factors, using real municipal wastewater. The results showed when C/N ratio was appropriately decreasing influent flow mode could achieve enhanced nitrogen removal with less adding of external carbon source, compared with equal influent flow mode. Three-step equal influent flow mode was recommended to treat common municipal wastewater in view of operation complexity. Non steady-state experiments over about three months confirmed step-feed SBR was an enhanced nitrogen removal process with high efficiency. Total nitrogen in the effluent was under 2 mg/L, the average removal efficiency achieved for TN was over 97% just adding a little external carbon source, and good sludge settleability was obtained.

[Effects of influent C/N ratio on performance of UniFed SBR process].

The effects of 7 groups of different influent C/N ratio (2.75, 4.28, 5.7, 6.5, 6.97, 8.08, 11.19) on the removal efficiencies of total nitrogen, phosphorus and COD as well as the sludge settling performance were investigated in a UniFed SBR lab-scale apparatus treating actual domestic wastewater. The results showed that when the C/N ratio was lower than 5.7, TN removal efficiency increased sharply as C/N ratio increased, from 43.6% of 2.75 to 80.84% of 5.7. Afterwards, TN removal efficiency increased very slowly as C/N ratio increased, because only TN removal efficiency caused by SND during aeration period could be enhanced. P removal efficiency increased as C/N ratio increased, because PAOs could get more organic carbon source from the influent to produce PHB and release PO4(3-), avoiding the adverse influence of NO(x)- on PO4(3-) release. The higher the C/N ratio, the more the quantities of P release and uptake, the quicker P uptake rate, the better P removal performance was. Regardless of the C/N ratio, excellent COD (average 93.15%) removal efficiencies were maintained throughout the experiment. When Influent C/N ratio was bigger than 6.97 and organic load exceeded 0.38 kg/(kg x d), SVI rose quickly as C/N ratio increased. Non-silk bacteria sludge bulge appeared because of low DO and high organic load.