Noval Identification Of SIRT1 Interaction Proteins Based On Yeast Two-hybrid System And The Regulation Of MICU1 By SIRT1

With the aging of the population in China tends to severe,up to now the country over 60 years old has a population more than 200 million.Accompanied by a variety of aging related diseases,malignant tumor,cardiovascular disease,neurodegenerative disease(Alzheimer's,Parkinson's disease and so on),cerebrovascular disease(stroke),diabetes and other aging related diseases not only seriously affecting the life of the aged,but also bring a heavy financial and spiritual burden to the family and society,the harmonious stability severely hinder the national economic development.Therefore,studying the pathogenesis of aging related diseases for the treatment of aging related diseases,and even delay the senescence to prevent diseases is the important topic in the medical science research in our country.The aging of people affected by multiple factors,the adult human organs tends to be stable,only a few organs with partial regeneration function,by the interaction of environment and its genetic factors,the body produced more and more gene mutations and inflammation,eventually leading to cell metabolism abnormality and dysfunction of organs.There are many aging related genes,such as SIRT1 which plays an important role,studies have shown that SIRT1 can prolong the life of the yeast[1],also has life prolonging effects in animal[2].Further studies showed that SIRT1 is involved in the process of a variety of cellular activities,works as a regulator of gene silencing,regulate gene expression through Foxo family involved in cellular oxidative stress and autophagy[3,4],influencing apoptosis of cells through P53[5],as a tumor suppressor reverse regulator of c-myc and mTOR pathways[6],acts in the hypothalamus to influence the biological rhythm and feeding behavior[7-10].In short,SIRT1 has many biological functions in regulating the cell,in order to know more about SIRT1,it is necessary to find out more protein that interacts with SIRT1.We used the classic method of interaction proteins screening,yeast two hybridization,according to the demand we construct the bait fusion protein BD-SIRT1,and to detect the yeast strain Y2HGOLD that contain BD-SIRT1 with no toxicity and self activation phenomenon.Human brain tissue cDNA library was bought from Clontech for hybridization.We finally screened out 27 proteins that may be interacting with SIRT1.We need selected several proteins from these 27 proteins to be verified and at last choosing one protein for further biological function research.The 27 proteins include transcription factors,mitochondria related proteins,lysosomal associated protein,vesicle associated protein and translation related protein etc...Because mitochondrial works as cell energy factories,in cell senescence the process of pathological changes is often accompanied by abnormal mitochondrial function,including neurodegenerative diseases such as Alzheimer's,Parkinson.In the process of cerebral ischemia-reperfusion which mitochondrial function failure often accompanied by calcium overload and it will cause excessive mitochondrial superoxide then induce cell death.So studying the relationship between SIRT1 and mitochondria,become the focus of our research.We have got five mitochondria related candidate proteins:DCTN6,ATP5F1,MICU1,SLC25A5 and MFF.After studying the existing reports,we focus on MICU1.MICU1,mitochondrial calcium uptake protein 1,the main function is to regulate mitochondrial calcium channel MCU,enable the cells to establish the appropriate level of calcium uptake in normal circumstances,to maintain the stability of cell mitochondrial metabolism,prevent the generation of reactive oxygen species under excess calcium overload.Research shows that in the MICUJ1 stable knockdown cells,the cells will produce higher concentration of Ca2+;so that cells maintain in high oxidative stress status.We know that most of' the time activation of SIRT1 has the protection effect of cells,otherwise inhibit the SIRT1 will make the cells more susceptible to apoptosis,so whether SIRT1 protect mitochondrial calcium homeostasis through MICU1 is unknow,and we will check this in our next studies.In order to verify the reliability of SIRT1 and MICU1 interaction,we performed experiments in vitro:first we transfected Hela cells with plasmid MICU1-HA,and then use the PBS buffer containing protease inhibitors to produce cell lysate,construction of GST-SIRT1 fusion protein to do GST pull-down experiment.Western blotting experiment showed that SIRT1 and MICU1 are interacting in vitro.In the in vivo study,Hela were spectively transfected with SITR1-flag,MICU1-HA,SIRT1-flag+MICU1-HA,SIRT1-flag+MICU1-HA plasmids,and then used IgG or falg antibody for immunoprecipitation,we got the result shows that SIRT1 and MICU1 have interaction in Hela cells.By immunofluorescence co-localization experiment,under the confocal microscope we can observe that MICU1-HA and SIRT1 exists co-localization.We used protein sample from cultured cortical neurons of rat to do immunoprecipitation experiment,the result also showed that the interaction between SIRT1 and MICU1.Consider above results,we think that SIRT1 and MICU1 interaction exist in vitro and in vivo.What functions the interaction between SIRT1 and MICU1 will have?We know that MICUl's main function is to regulate mitochondrial calcium uptake,does SIRT1 regulates mitochondrial calcium intake through MICU1?We first observe inhibition or activation of SIRT1 activity and its effects on mitochondrial calcium uptake.Using DMSO as control,10?M of resveratrol as a SIRTI agonist,lOmM nicotinamide(NAM)and 10?M EX527 as SIRTI inhibitors,respectively pre-treated cells with 1 hour,then incubated mitochondria calcium dye Rhod-2/AM half an hour,finally washed out the dye and observed under confocal laser scanning microscopy to calculate fluorescence intensity.Recording 1 minutes'data under the basal condition and then using 100?M histamine to induce mitochondrial calcium influx.Observation results showed no significant differences between the groups(DMSO 33.647±10.93%,NAM 39.553±16.89%,EX52746.077±19.73%,Resveratrol 32.73±11.37%)in calcium signal level before applied with histamine,this reminded us that inhibition or activation of SIRTI under the normal condition had no effect on the activity of mitochondria calcium uptake,but in the histamine induced calcium influx case(DMSO 107.78±9.36%,NAM 173.27±7.31%,EX527 185.92±17.44%,Resveratrol 122.29±17.58),with SIRTI inhibitors,the group NAM(p<0.05)and group EX527(p<0.05)significantly increased inward calcium current amplitude compare with DMSO control group.This indicated that the inhibition of SIRTI resulted mitochondrial lost the ability to control the large influx of calcium in normal condition.Further more,we used shRNA to knockdown SIRT1 in Hela cells.There is significantly increased inward calcium current amplitude in SIRTI knockdown group(90.295±2.83%)compare with shNC control group(90.295±2.83%)(p<0.05).So we can draw the conclusion that SIRTI can influence the calcium uptake of mitochondria,at least the calcium overload under the influence by external factors will be enhanced by the inhibition of SIRTI,and this let the cells more susceptible to damage.With the current studies,they're consistent with above conclusion that in most cases the lethal effect on cells of inhibition of SIRT1 activity.Then the effect of SIRT1 on mitochondrial is it work through MICU1?In order to verify this issue,we express plasmid of MICU1-HA and PCMV-HA in Hela cells.The two group of cells is 1 hours ahead treated with 10?M EX527,and then detected the mitochondrial calcium concentration used confocal microscopy,results showed that over expression MICU1-HA group has no significant difference with PCMV-HA group before applied with histamine,indicated that over expression of MICU1 in normal circumstances has no effect on the level of mitochondrial calcium uptake,it's consistent with the literature reports.Then,after induced by histamine,the maximal concentration of calcium in MICU1 over expression group(90.66514.78%)generated was significantly lower than the control group(157.81913.87%)(p<0.05);this showed that calcium overload enhancement phenomenon produced by the inhibition of SIRT1 is partially mediated by MICU1.SIRT1,as NAD(+)-dependent protein deacetylase,is an important regulator in cellular stress response and energy metabolism.,mainly through deacetylate other proteins to carry out its function.Could MICU1 be one of SIRT1's deacetylate target?We express MICU1-HA in Hela cells for 24hours,and then treated cells with DMSO or 10 ?M EX527,using HA antibody to immunoprecipitate MICU1 protein.Western blot reveal that there is no change of acetylation level between two groups.To sum up,the SIRT1 can be directly interacts with MICU1,and inhibit the activity of SIRT1 can enhance the strength of mitochondria calcium overload via the MICU1,this will make the cell more easy to be calcium overload.Our findings reveal a novel mechanism for SIRT1 carry out its duty,which may be help with finding new drugs.