Mechanisms Of Acetylation Modification Of ANXA1 Decreasing Cellular Apoptosis After OGD/R Injuries

BackgroundIschemic stroke（IS）is the main course of neuronal cells damage resulting in ischemic/reperfusion（I/R）,and the cellular apoptosis is one of the elementary mechanisms of I/R injuries in IS.Hence the scientific researchers attach the most importance to elucidate the implied apoptotic mechanisms of neuronal cells in IS process.The previous studies form the author’s colleagues indicated that annexin a1（ANXA1）has participated in the regulation of neuronal apoptosis induced by I/R injuries.The molecular effects of ANXA 1 in IS depends heavily on its association with the other proteins and its protein modifications,as well as its subcellular distributions.The previous studies of the present scientific research group demonstrated that under I/R stimulations,the phosphorylation level of ANXA1 has increased to promote its nuclear translocation and increase transcript level of the pro-apoptotic factor bid to induce cell apoptosis.The research in the SUMOylation of ANXA1 show the SUMOylation of ANXA1can enhance selective degradation of IKKαin microglial cells to reduce the pro-inflammation factors production induced by OGD/R and protect neuronal dysfunctions of mice injured by I/R process.Except for phosphorylation and SUMOylation,ANXA1 can be acetylation modified.Recent scientific studies report the deacetylation effects of SIRT2 to the target proteins is responsible for the aggravation of the neuronal defects and increasing neuronal cells apoptosis in the animal mode of IS.If ANXA1 can be acetyl modified under I/R condition?Or if the acetylated ANXA1 can induced the neuronal cells apoptosis resulting of I/R injuries?To answer these questions,OGD/R cellular mode and MCAO/R animal mode of I/R is introduced to this research,as well as establishing the acetyl mimic plasmid of K312QANXA1 and the acetyl silent plasmids of K58R ANXA1,K71R ANXA1 and K312R ANXA1 to study the molecular effects and mechanisms of apoptosis induced by acetyl modifications of ANXA1 after I/R stimulations.Material and methodsCell mode of OGD/R and animal mode of MCAO/R are used to simulate I/R injuries caused by ischemic stroke.Acetyl-mimic of K312Q and acetyl-silent of K312R ANXA1plasmids are generated by site-mutagenesis technic.IP is to test the acetyl level of ANXA1and CO-IP is used to test the associations of the proteins regulating by acetylated ANXA1.WB and RT-PCR are utilized to detect the changes of proteins and m RNA resulting in the acetyl changes of ANXA1,and IF is to detect the colocations of two proteins.OD405nm is used to assess the absorbance of substrates cleaved by CASP3 and CASP9.Proteomic studies analyze the expression difference,GO enrichment and KEGG pathway enrichment.Results1.The binding of ANXA1 and SIRT2 is increasing and the acetyl level of ANXA1is decreasing under OGD/R or MCAO/R conditions,but this effect is converted by pretreating with SIRT2 inhibitor AGK2.To exemplify the interrelationship between ANXA1 and SIRT2,HEK293T cell was treated with OGD/R,CO-IP result indicates that compared with the control,the binding of ANXA1 and SIRT2 is increasing and the acetyl level of ANXA1 is decreasing correspondingly after OGD/R.And the acetyl level of ANXA1 is further declining after overexpressing the GFP-SIRT2 plasmid into HEK293T cells after OGD/R induction.Conversely,the cells pretreated with SIRT2 inhibitor AGK2 can turn over the acetyl declination of ANXA1 induced by OGD/R.Compared with the OGD/R group,acetyl level of ANXA1 in AGK2+OGD/R cell is increasing significantly.Subsequently,mice mode of I/R is established with MCAO/R to observe the binding of ANXA1 and SIRT2,then to evaluate the acetylation change of ANXA1.IF results present high accordance with the CO-IP,that ANXA1and SIRT2 colocate on hippocampus in MCAO/R mice.IP result shown that the acetyl level of ANXA1 in the ipsilateral side of MCAO/R mice is decreasing than that of the contralateral side drastically.Pre-injection of AGK2 into the caudal vein can convert the decreasing acetyl level of ANXA1 induced by MCAO/R and the acetyl level is elevating significantly accordingly.Generously,the acetyl level of ANXA1 is decreasing due to the enzyme activity changes of SIRT2 under OGD/R or MCAO/R condition.2.OGD/R stimulation can enhance nuclear translocation of ANXA1 but acetyl modified ANXA1 or pretreating with AGK2 can limit ANXA1 translocating into the nuclear but increase its recruitment in the cytosol.To clarify the influence of acetyl level changes of ANXA1 in the subcellular distributions after OGD/R,AGK2 pretreatment is introduced into the I/R cell mode to access the subcellular locations of ANXA1 in cytosol and nuclear.Western blot results show that OGD/R induces ANXA1 translocation into nuclear and minimize its cytosol distribution compared with the control group.Pretreating with AGK2 to limit SIRT2 activity in the HEK293T cells can convert the subcellular dislocation of ANXA1 caused by OGD/R stimulation.Compare with the group only treated by OGD/R,the nuclear translocation of ANXA1 in AGK2+OGD/R group is decreasing significantly,and recruiting in the cytosol drastically.Screening and blasting the databases of the Uniprot Protein Resource and Phosphosite Plus,K58,K71 and K312 residues are selected as potential target acetyl sites of ANXA1.The acetyl-silent mutants of GFP-tagged of K58R,K71Rand K312R ANXA1plasmids are generated to determine which lysine residue was the target site of SIRT2affecting on ANXA1.CO-IP is utilized to observe the interaction between acetyl sites of ANXA1 and SIRT2.The results show that the K312R mutant of ANXA1 is the least binding with SIRT2,therefore K312 was selected as the target site of SIRT2 effecting on ANXA1.Then the acetyl mimic mutant of K312Q ANXA1 is built,HEK293T cells are overexpressed with WT ANXA1,K312Q ANXA1 and K312R ANXA1 to evaluate the subcellular distribution of these acetyl mutants after OGD/R.Compared with WT ANXA1,K312Q ANXA1 has accumulated significantly in the cytosol and decreased in the nucleus after OGD/R.3.Pretreating with AGK2 can promote the defective signs of neuronal dysfunctions and restore abilities of learning and memory in mice of MCAO/R.Neuronal deficiency scores,Rotarod test and MWM are utilized to test if AGK2preinjection can enhance the neuronal and learning dysfunction in mice injured by MCAO/R.The neuronal deficiency scores indicate that the neuronal injured signs in mice of MCAO/R is worse than that of sham operated mice.But AGK2 pretreating mice of MCAO present significant promotion in neuronal deficiencies scores than that of mice who were treated merely with MCAO/R.There exists highly consistency of results in the Rotarod test in which the fallen latency of AGK2+MCAO mice is much longer than that of DMSO+MCAO mice.MWM is used to test the abilities of space orientation and memory of mice,it can be observed that the escape latencies in AKG2+MCAO group are shorter more than DMSO+MCAO group in 6 d training test and the 7th d probing test.The frequency of crossing the platform,the latencies and the distance of Q1 zone present more excellent in AKG2+MCAO group in the probing test.4.Overexpression of K312Q ANXA1（acetyl-mimic of ANXA1）can inhibit activities of apoptotic relevant protein CASP3 and CASP9 induced by OGD/RTo further explore the influence of acetyl changes of ANXA1 on cell apoptosis after OGD/R,proteome data are used to analyze the different expressions of apoptotic relevant proteins in HEK293T cells overexpressing WT and K312Q ANXA1 after OGD/R.The results from GO data analysis indicate that the difference of protein has involved in the mitochondrial pathway,and proteins of regulating apoptotic program.The analysis on KEGG pathways also suggest that the relevant apoptotic enrichment in multiple species.These results demonstrate that K312Q ANXA1 is an elementary factor in regulation of apoptotic program on the cellular processes,especially in the mitochondrial pathway.To clarify the influence of acetyl modified ANXA1 on apoptotic proteins after OGD/R,Western blot and OD405nm absorbance are used to test expression and activities level of CASP3 in HEK293T cells overexpressing WT,K312Q and K312R ANXA1.Western blot results show that compared with WT and K312R ANXA1 groups,the active form of CASP3,cleaved CASP3 is significantly decreasing in K312Q ANXA1 after OGD/R.And the absorbance of substrate of CASP3,Ac-DEVD-p NA is declining drastically in K312Q ANXA1 overexpressed cells after OGD/R.As the upstream CASPs to cleave CASP3,the OD405nm absorbance of CASP9 induced by OGD/R are minimizing in K312Q ANXA1transfected cells comparing with WT ANXA1 after OGD/R.Meanwhile the cleaved form of CASP9 is decreasing correspondingly,but expression of t Bid is not changed anyhow in the cell overexpressed K312Q ANXA1 after OGD/R.These experiments demonstrate that K312Q ANXA1 has potency to reduce cell apoptosis induced by OGD/R damage.5.K312Q ANXA1 can downregulate protein level of regulate subunit of PKA,PRKAR2B to enhance kinase activity of PKA and promote the serine phosphorylation of CASP9If there is no relationship between decreasing cleaved CASP9 and protein amount of its upstream t Bid,it is necessary to further explore the modification of CASP9 associating with its cleavage.Proteomic analysis is performed in HEK293T cells overexpressing WT ANXA1 and K312Q ANXA1 under OGD/R condition.The results of proteome show one of the upstream kinases of CASP9,PRKAR2B is downregulating.But the m RNAs of PRKAR2B and PRKACA have little changes before and after OGD/R treatment.Western blot results show comparing with WT ANXA1,K312Q ANXA1can decline protein level of PRKAR2B after OGD/R.The explain for these results is that the effect of K312Q ANXA1on PRKAR2B downregulation on protein level and not on transcriptional level after OGD/R.Downregulation of protein PRKAR2B can enhance kinase activity of PKA and promote serine phosphorylation of CASP9 under OGD/R condition.6.After OGD/R,excessive accumulation of K312Q ANXA1 in the cytosol initiates the autophagy pathway and degrade PRKAR2B who binding with K312Q ANXA1 via autophagyConsidering that previous studies showed the protein level of PRKAR2B was downregulating after OGD/R,the protein of PRKAR2B might be degrade via autophagic mechanism.Analyses from proteomic data of autophagic protein and autophagic relevant factors indicate comparing with WT ANXA1,overexpression of K312Q ANXA1 can upregulate AMBRA1 level in HEK293T cells after OGD/R.Western blot results indicate that AMBRA1 and the biomarker of autophagy LC3Ⅱis increasing.Pretreating the cell with autophagic inhibitor NH₄CL can invert the decreasing PRKAR2B protein in cell overexpressed with K312Q ANXA1 after OGD/R.Therefore,it is K312Q ANXA1 which accumulating excessively in the cytosol to trigger the autophagy pathway and mediate degradation of PRKAR2B after OGD/R.ConclusionUnder condition of OGD/R,acetylation level of ANXA1 is decreasing significantly.Through downregulation of the regulatory subunit of PKA PRKAR2B,the upstream kinase of CASP9,can enhance catalyze activities of PKA to improve the serine phosphorylation of CASP9 and inhibit the cleavage and activation of CASP9 and CASP3 after OGD/R,consequently to minimize cell apoptosis induced by OGD/R injuries.