The Expression Of Selenoprotein P In Tumors And The Function Of Its Isoform Mutant Short Peptide Hsel P82m On Apoptosis

In addition to the wide recognition that Selenium(Se)is a necessary trace element with various bio-functions, some recent researches have revealed that it is negatively correlated with the occurrence of malignant tumors, especially with the gastroenterological and respiratory tract tumors. Se plays its bio-function through selenoprotein, of which Selenoprotein P is a special isoform with 10 Se-cysteines. Se-cysteines are usually encoded by the terminal codon UGA in the coding frame, so its mechanism of synthesis and incorporation with protein is complicated. Since no successive ways have been set up for the recombinant expression of selenoprotein until now, and its purification from plasm is too tedious to maintain the activity, further investigation to clarify the characteristics and functions of selenoprotein is certainly required.Our study consisted of four parts. In part I , the expression and distribution of Selenoprotein P was analyzed with the polyclonal antibody prepared by our group first in gastric, colon and liver cancer tissues, then in 4 types of hepatic cancer cells. The results showed that allthe cancer tissues expressed Selenoprotein P, but at a significantly lower level compared with the normal tissues, and hepatic cancer cells also expressed Selenoprotein P. The expression of Selenoprotein P was found to be related to tumor grades in gastric and colon cancers except hepatic cancer, but not relevant to the TNM stage of all cancer tissues. The study demonstrated that the low expression of Selenoprotein P as a protective factor may be correlated with the occurrence of tumors.Two isoforms of Selenoprotein P, mutant short peptide hSel P280m (280 aas) and hSel P137m (137 aas) , together with their antigen peptide (all prepared in our lab), have been proved biologically active. In part II, in order to analyze the apoptosis- inducing domain of Selenoprotein P mutant peptides, to find apoptosis- inducing short peptide, and to screen an effective and safe apoptosis inductor, we made further analysis of hSel P137m. We found two cleavage sites of hydroxylamine at 27/28a and 109/110a respectively in its primary structure, with 82 amino acids between the two sites. In the primary structure of the 82-aas peptide, we found Casein kinase II phosphorylation site, Protein kinase C phosphorylation site and N-glycosylation site, and three important domains: FHA, PDZ, SH2. With bioinformatic theory and methods, the SelP-N domain ( SeCys^40thas the center) was showen as the key part of the activity of the peptide. Then hSel P137m was cut with hydroxylamine, and its split product was obtained with the SelP-N domain remained, named hSel P82m. In the study, we found hSel P82m could inhibit and kill hepatic cancer cells, which indicated the superior bioactivity of hSel P82m.More and more evidences showed that mitochondrial related withapoptosis. The opening of mitochondrial permeability transition pore(MPTP) can cause the release of the ions, solute micromolecule and some proteins of mitochondrial matrix. Then ion gradient disappears between two sides of endomembrane, causing the decrease or collapse of transmembrane potential (ΔΨm). After the ΔΨm collapses, the mitochondria will release cytochrome C(Cyt C), apoptosis inducing factor(AIF), Ca²⁺ ,reactive oxygen species (ROS), and so on. The release of Cyt C can cause the blockage of the oxidative phosphorylation, exhaustion of ATP, increasing of ROS, then induce the necrosis, or activate Caspases to induce apoptosis. So, the opening of MPTP and the collapse of ΔΨFm are considered the special markers of cell apoptosis. In part III, with the mitochondria of mice liver in open system, we found that hSel P82m could cause the opening of MPTP and the collapse of ΔΨFm, then result in the release of Cyt C. The study indicates that hSel P82m can promote the cell apoptosis correlating with the opening of MPTP and the collapse of ΔΨm, and the release of Cyt C.Peptides are involving various cell functions as the bioactivity substances. The segment neighbouring SeCys^40th is the key part of the active domain of hSel P280m, hSel P137m and hSel P82m. We hypothesized that we could find and ascertain an apoptosis-inducing short active peptide with the SeCys^40th as the center, based on the hSel P82m. Then we could screen a safe apoptosis inductor with high efficiency targeting MPTP. In part IV, on the prophase work, we designed and synthesized artificially a 15aas peptide containing N-terminal domain of Sel P. The 15aas peptide could cause the opening of MPTP and the collapse of ΔΨm, though in a weaker manner...