Study On The Impact Of Transmembrane Protein 21 γ-Secretase Involved In Alzheimer's Disease

Objective Previous studies have revealed thatγ-secretase is involved in the pathogenesis of Alzheimer's disease.The impacts of the transmembrane protein 21 (TMP21) on the protein levels ofγ-secretase components and theγ-secretase activity were investigated in APP knockout mouse embryonic fibroblasts(MEF(KO)).The expression of TMP21 in MEF was inhibited by siRNA transfection or overexpression by cDNA-TMP21,and the blank plasmid was used in the control. During the purification ofγ-secretase,samples comprisingγ-secretase complex were collected in transfection(T) or control(C) group at three phases.The samples were respectively marked as follow:T1 and C1 which included the whole cell membrane proteins;T2 and C2 which were the lysates by CHAPSO;IPT2 and IPC2 which were prepared after co-immunoprecipitation by presenilin-1 antibody.The protein levels of TMP21 andγ-secretase components,presenilin-1(PS-1) and Nicastrin(NCT),were dectected by Western blotting.Theγ-secretase activity against artificial substrate C99 was measured by ELISA.The major results are as follows:1.Take a new-born rat brain,deliquescenced transgene rat brain by CHO and PS1-/-Cells line to centrifugalize,incubation and others measures to get APP-CTF (C-terminal Fragment of APP).Then to study Characterization of Cytoplasmic C-terminal Fragment of APP by IP(Immunoaffinity) Gel-HPLC(Gel-filtration -HPLC),RP-HPLC,N-terminal sequencing and MS(mass spectrometry).The study showed that Mechanisms of the intramembrane cleavage on the C-terminal fragments of APP maybe isγsecrtases andεsecrtases cleavage C-terminal fragments of APP.Maybe besideα,β,γ,there isεsecretases.2.The expression difference of the rat brain and the HEK293 cells line.then Analyze with Western Blotting.the result showed that TMP21 must be take place a function with one of compositions withγ-secretases,Repress a function to Aβ40and Aβ42's acticity.But the Concretely combine district not clear.That is to be proven in the further research.3.The research of set up the interference vector of TMP21 and succesed in set up interference vector in cells molde.The researchers find that when make TMP21 interference,the PS1 complex are effected grestly and Aβ40 productions are increase.4.The protein levels of TMP21 in T1,T2 and IPT2 were,respectively,decreased by 14.8±2.1%,46.5±4.7%and 68.9±3.4%over C1,C2 and IPC2.Meanwhile,no change in the expression levels of NCT and PS-1 was observed.The amounts of Aβpeptide40 and Aβpeptide42 from C99 catalyzed byγ-secretase in T1,T2 and IPT2 were increased to 13.9%(p = 0.24),87.2%(p＜0.001),211.4%(p＜0.001) when respectively compared to C1,C2 and IPC2.The activity ofγ-secretase was highest in IPC2 in the comparison with C1 and C2;indicatingγ-secretase was purified after sample treatment.5.To check the production of PS-1,NCT and TMP21after overexpression TMP21 assay by Western blotting.Use ELISA check the activity ofγ-secretases and dataprocessing with statistics.It is showed that the activity ofγsecretases increase more as no other factors change the TMP21-overexpression.6.From above research suggest that TMP21 could be one of the components ofγ-secretase complex.The activity ofγ-secretase was increased in MEF by the inhibition of TMP21 expression via siRNA transfection or overexpression via cDNA-TMP21.Therefore,transmembrane protein 21 can be one of the negative modulators ofγ-secretase responsible for Alzheimer pathogenesis.