The Mechanism Of E3 Ubiquitin Ligase P53RFP Mediated HIF-1α Degradation In Prolonged Hypoxia

BackgroundsIschemic diseases (coronary atherosclerotic heart disease, ischemic cerebrovascular disease and peripheral vascular ischemic disease) have become a serious impact on human health. Therefore, it became necessary to explore new treatment methods and ways to treat ischemic diseases. In recent years, therapeutic angiogenesis for the treatment of ischemic diseases provides a new direction. In therapeutic angiogenesis, the choice of different stages on angiogenesis of combined use of several factors or sequential treatment, or select the main trigger of angiogenesis may be more effective. HIF-1α (hypoxia-inducible factor-la) is a for upstream key transcription factor, when cell is adaptive response to hypoxia or anoxia. It has bee confirmed to induce over 70 transcription factor. It can adjust the angiogenesis and create a new way of thought for reascularization treatment. Therefore, HIF-1 a, as a application prospect of therapeutic factors, in the field of medical research has more attraction and attention.It has been clear about the sites of the structure and the regulation of HIF-1 a, it consists of HIF-1 alpha and beta two subunits. Between them, the alpha subunit is the key for adjustment unit for the biological function of HIF-1, its protein stability and transcriptional activity are highly regulated by the oxygen concentration. The composition of HIF-1 beta is only for structure. Under the condition of hypoxia, HIF-1 a expression level increased, and formed dimers with HIF-1 beta, thus has the biological function. HIF-la stability and activity is adjusted by the Oxygen concentration, because of HIF-1 alpha containing Oxygen Degradation area (Oxygen Dependent Degradation Domain, ODDD) and transcriptional activation area (Transactivation Domains). In constant condition, Pro402 in ODDD area and/or Pro564 are hydroxyled by proline hydroxylase (prolyl hydroxylase domain, PHD), and identified by VHL (von Hippel Lindau tumor suppressor) tumor suppressor protein complex, degradation by ubiquitin proteasome pathway. At the same time, factor inhibiting HIF-1α (FIH) combined with HIF-1α C terminal transcription activation zone (C-TAD), by 803 loci asparagine residues (Asn803), hydroxyl, prevent C-TAD combine with auxiliary activation factor (CBP/p300), thus inhibited the transcription activity. In previous studies, we successfully mutanted alpha the 564, 402,803 three loci joint point of HIF-la, and constructed three mutant HIF-la Triple expression vector (pcDNA-(3.1+)-Triple-HIF-1α), make it can express stably and efficiently under constant oxygen, which is essential for the study of HIF-la gene function in the foundation experiment.p53RFP is a newly discovered an important genes regulating cell proliferation and apoptosis, under the regulation of p53 gene, its product has the E3 ubiquitin ligase activity, and is an important start signal degradation by the ubiquitin-proteasome apoptosis p21WAF1 involved in cell proliferation and apoptosis, which involved in cell cycle regulation. At present the research of the gene is not much. We found in previous studies, when HIF-la expression increased, p53RFP gene expression is upforward. And as an E3 ubiquitin ligase, it is unknown whether p53RFP can specificly indentify HIF-1α and start HIF-1α ubiquitin degradation pathway, as well as the possible mechanism.ObjectiveThis study is proposed to verify whether p53RFP has a relationshippand with the degradation of HIF-la, to explore the possible mechanisms and channels. These could be the fundamental for the further intervention research, which makes HIF-la play a positive role in the ischemia hypoxia environment.Method1. SW480 cells are cultured under hypoxic incubator (1% O2,94% N2 and 5% CO2). At the different time, HIF-la and p53RFP protein expression levels are detected, to identify whether there is degradation process f two proteins in the prolonged hypoxia, and the possible relationship between them.2. Add protease inhibitors MG132, to identify whether HIF-la degrade through ubiquitin proteasome pathway in prolonged hypoxia.3. Through the interference test, blocking and promoting p53RFP expression in cells, confirm whether the degradation of HIF-1α in prolonged hypoxia is associated with p53RFP.4. Through RT-PCR method, to confirm whether p53RFP has influence on the transcription level of HIF-1α.5. Under the condition of constant oxygen, through three mutant HIF-la eukaryotic expression vector, to prove whether the control function of p53RFP on the degradation of HIF-la is dependent on VHL.6. In prolonged hypoxia, through the application of p53 inhibitors, to explore whether the control function of p53RFP on the degradation of HIF-la is dependent on p53.7. By Co-IP, to identify whether p53RFP combine withe HIF-la through ubiquitination, then play a promote role of HIF-la degradation.8. In prolonged hypoxia, the application of histone acetylation enzyme inhibitors, to explore whether the control function of p53RFP on the degradation of HIF-la involve histone acetylation enzyme HDAC1 and HDAC2.9. In prolonged hypoxic, the transition of HDAC1 SiRNA and HDAC2 SiRNA, to further explore histone acetylation enzyme HDAC1 and HDAC2 in p53RFP degradation of HIF-la, whether associated with the degree of acetylation of HIF-la.ResultsThe first part:In hypoxia, HIF-la protein levels began to increase at first, and reach its peak at 6 hours, in the following hours of 12,18,24, after HIF-1α degraded, eventually maintained at the low expression level. In the detection of endogenous p53RFP protein expression, it is found that as the extension of incubation time in hypoxia, p53RFP expression also have certain extent increase.After adding protease inhibitors MG132, the expression of HIF-la rised obviously. Different concentrations of protease inhibitors MG132 blocking HIF-la degradation has different effects, it is more obvious with the increase of the concentration.The second part:Compared with hypoxia treatment group 24 hours alone, the intracellular HIF-1α protein degradation of transient transfection exogenous p53RFP plasmid (1,2, and 6 ug) cells is more apparent, and the expression of p53RFP quantity is higher, which promote HIF-1α protein degradation more obviously. The intracellular HIF-1α protein degradation of transient transfection exogenous p53RFP SiRNA (976) 1,2,6 ug cells is less obvious, and the expression of p53RFP quantity is lower, which reduce HIF-1α protein degradation more obviously.Under the condition of constant oxygen and prolonged hypoxia, transfection p53RFP or not has no obvious effect on the mRNA level of HIF-1α.The third part:Compared with pcDNA 3.1 (+)-HIF-1α triple group, the HIF-1α expression of p53RFP and pcDNA 3.1 (+)-HIF-la-triple transfection group to decrease obviously, and degradation increased, meanwhile p53RFP expression increase. Compared with whether adding the p53 inhibitors, the expression of p53RFP and HIF-1α of p53RFP transfection group has no obvious change, and p53RFP rise obviously, HIF-1 alpha decreased obviously compared with no interference factors group.As the extension time of hypoxia, with the degradation of the protein levels, the acetylation and ubiquitin modification levels of HIF-1 are increased.Transient transfection p53RFP plasmid 1,2,6 ug, then hypoxic training for 24 hours, it showed that in low oxygen conditions p53RFP can combine with HIF-la directly, and promote its ubiquitination modification and degradation.Compared with whether adding the histone acetylation enzyme inhibitors, the expression of HIF-1α of P53RFP transfection group decreases obviously.Compared with whether transiting HDAC1 SiRNA and HDAC2 SiRNA, the expression of HIF-la of P53RFP transfection group increases, and acetylation of HIF-1α is lower obviously.ConclusionThe first part:1. In prolonged hypoxia, the expression of HIF-1α gradually rose and peaked at 6 hours, then it degraded and maintain at a low level.Under the same conditions, p53RFP expression gradually increased.2. In prolonged hypoxia, the degradation of HIF-1α is through the ubiquitin-proteasome pathway.The second part:1. In prolonged hypoxia, after transfection of p53RFP plasmid, p53RFP expression increased, promoted HIF-la degradation, p53RFP expression and the degradation of HIF-1 alpha is positive correlation.2. In prolonged hypoxia, after transfection of p53RFP SiRNA plasmid, p53RFP expression reduced as well as HIF-1α degradation, it confirmed that p53RFP expression and the degradation of HIF-1 alpha is positive correlation.3. In constant oxygen and hypoxia, p53RFP has no effect on HIF-1α transcription level.The third part:1. P53RFP can promote the degradation of HIF-1α three mutations.2. In prolonged hypoxia, p53RFP participation of HIF-1α degradation is VHL and p53 dependent.3. In prolonged hypoxia, p53RFP can combine with HIF-1α directly, and promote its ubiquitination modification and degradation.4. In prolonged hypoxia, p53RFP participation of HIF-1α degradation is negatively regulated by histone acetylation enzyme HDAC1 and HDAC2.