| ABRO1, also known as the KIAA0157or FAM175B, belongs to the FAM175family and it is ABRA1brother protein, so called ABRO1. At present, we know very little about its function. A recent study shows that ABRO1participates in BRISC complex (BRCC36-containing isopeptidase complex) formation. BRISC complex formed by BRCC36,BRE,NBA1and ABRO1, specifically cleaves Lys63-linked polyubiquitin chains. ABRO1as a central scaffold protein promotes BRISC complex assembly and catalyzes the deubiquitinating enzyme activity. Recently, ABRO1is reported to interacts with zinc finger transcription factors THAP5which control the G2/M cell cycle and apoptosis. In myocardial cell, overexpressing ABRO1significantly reduces the Lys63-linked polyubiquitin chains of specific target protein and reduces the cell damage and cell death, which has an important role in myocardial infarction of coronary artery disease(CAD) patients and myocardial protection. In addition, early to the ubiquitin-proteasome enzyme and the proteomics analysis of related protein complex shows that USP7may combine with ABRO1. ABRO1related to p53function is not reported. The laboratory work shows that ABRO1expression significantly reduces in a wide variety of tumor tissues. Therefore, we carry out a deep research on ABRO1biological function and mechanism based on the early work of our laboratory.In this study, we first prove that overexpressing ABRO1significantly suppresses HCT116p53+/+cell clone formation, but without obvious effect on HCT116p53-/-cell clones with cell cloned formation experiment by constructing HCT116p53+/+and HCT116p53-/-cell lines stably overexpressing or low expressing ABRO1. However, knockdown of ABRO1promotes HCT116p53+/+cell cloned formation, and not affects HCT116p53-/-cell clones. Then through the cytometric analysis we find that overexpressing ABRO1can induce HCT116p53+/+cells G0/G1phase block. Lude mose tumor-burdened experiments have confirmed the ABRO1can inhibit tumor growth, and have p53dependence. We use Co-IP and GST-pulldown experiments to show that ABRO1directly interacts with p53, and determine the interaction domain. Western blot experiments show that overexpressing ABRO1significantly prolongs the half-life of p53and knockdown of ABRO1significantly shorten its half-life. Overexpressing ABRO1can restrain MDM2mediated p53nuclear export, reduce the ubiquitin-proteasome degradation of p53; knockdown of ABRO1promotes p53nuclear export, increases the ubiquitin-proteasome degradation of p53. We use Co-IP and GST-pulldown experiments to prove the direct interaction between ABRO1, p53and USP7and they can form a stable protein complex without overlapped interaction sites. We use Co-IP experiments proved that overexpressing ABRO1can enhance the ability of USP7binding to p53and strengthen deubiquitination of USP7to p53; knockdown of ABRO1weakens the interaction between p53and USP7and increase the ubiquitin-proteasome degradation of p53. At the same time, we find that USP7does not affect ABRO1combined with p53, Co-IP experiments show that knockdown of USP7does not change the interaction between and p53. The body of the ubiquitination experiments show that knockdown of USP7weakens the ability of ABRO1to deubiquitinates p53; knockdown of ABRO1also weakens the ability of USP7to deubiquitinates p53.MDM2is the main E3ubiquitin ligase of p53, which mediates the ubiquitin-proteasome degradation of p53. MDM2and p53can respectively combine with the debiquitinating enzyme USP7, USP7can cleave both their poly ubiquitin chain; the affinity of MDM2binding to USP7is higher than p53. MDM2is a preferred substrate protein of USP7. We first overexpress ABRO1in HCT116p53-/-cells, finding that MDM2protein level is not significant changed. And then we first overexpress ABRO1in HCT116p53-/-cells and treat cells with CHX, finding that the half-life of MDM2do not changed greatly; Knockdown of ABRO1also does not change the half-life of MDM2. Co-IP experiments show that MDM2and ABRO1does not exist interactions in H1299cells. We use RNAi technology respectively to knockdown the NBA1, BRCC36and BRE of BRISC complex, and find that BRCC36does not affect p53protein stability but BA1and BRE can stabilize p53. However, western blot experiments show that knockdown of BRE and overexpressing ABRO1can still raise p53protein level. We also use Co-IP experiment to show that the USP7/p53/ABRO1complex and BRISC complex are not together.DOX can induce the DNA damage stress response. We use DOX to treat HCT116p53+/+cells, simulating DNA damage, and find the ABRO1protein expression increases with the DOX simulation extension; if we knockdown ABRO1, DOX stimulation cannot induce p53and p21expression rises. To further study, we transfect GFP-ABRO1, which mostly located in the cytoplasm, into the MCF7cells. With the DOX stimulation, GFP-ABRO1imports into nucleus. The DNA damage can increase HCT116p53+/+cell apoptosis, and induces G0/G1phase block. We use DOX stimulate HCT116p53+/+cells to induced DNA damage, and use RNAi technology to knockdown ABRO1. With flow cytometric detection cell apoptosis number, we find that knockdown of ABRO1significantly leads to apoptosis cell reduce, but there is no change in HCT116p53+/+; At the same time, knockdown of ABRO1leads to significantly reduce in the G0/G1phase cells, but there is no significant effect on the cell cycle of HCT116p53-/-cells.The laboratory work shows that ABRO1expression significantly drops in a wide variety of tumor tissues, especially in the liver cancer. Therefore, we use the liver tissue chip to examine ABRO1expression in157cases of samples, and the statistics analysis results show that ABRO1expression is lower in the liver tumor tissue than in its corresponding adjacent tissue. Furthermore, ABRO1expression has significant correlation with p53in liver tumor tissue, but there is no correlation between them in adjacent tissues. We also find that the patients with ABRO1positive expression survive longer than those with ABRO1negative expression after surgery.Our results show that ABRO1inhibits cell proliferation with the manner of p53dependence and we confirm that ABRO1can bind USP7and p53, forming a protein complex, to promote USP7and p53interaction, to deubiquitinate p53. The result is to increase p53stability and transcription activity, and block the G0/G1cell cycle. In the unstressed condition, ABRO1mainly locates in the cytoplasm. When DNA is damaged, ABRO1expression increases and imports into nucleus to participate in p53activation. Our experiments also prove that ABRO1regulates p53activity does not depend on BRISC complex activity and stability of MDM2. ABRO1expression reduces in liver cancer tissues, and its expression level in liver cancer tissues is related to the expression level of p53and the prognosis of patients. It reveals a new molecular mechanism of the down regulation of p53in tumor cells, and implies that ABRO1may be a new tumor suppressor and HCC prognosis testing index. In conclusion, our results suggest ABRO1is a new regulator of p53, plays an important role in regulating cell proliferation and DNA damage response.We assume that ABRO1distributed mainly in the cytoplasm, has continuous low expression in normal condition. This is helpful for USP7to target to MDM2and maintain p53at low level. Under genotoxicity stress, on the one hand, through the ATM dependent phosphorylation, the affinity of USP7and MDM2/MDMX become weak; on the other hand, DNA damage quickly induces increased ABRO1expression and import into nucleus, prompting USP7to target to p53, antagonisting the p53ubiquitination mediated by MDM2, stabilizing and activating p53rapidly. At present, we still don't know what mechanism is involved in the upregulation of ABRO1when DNA damages happen and whether there is any posttranslational modification to control ABRO1nuclear import.
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