The E3 Ubiquitin Ligase TRIM31 Is Involved In Cerebral Ischemic Injury By Promoting Degradation Of TIGAR

BackgroundStroke is an acute cerebrovascular disease caused by insufficient blood supply to the brain tissue due to stenosis or blockage of cerebral blood vessels.It can be divided into ischemic stroke and hemorrhagic stroke.It is the second leading cause of death among people over 60 years of age worldwide.Among this,ischemic stroke accounted for 77%of strokes.The main cause of cerebral ischemia is middle cerebral arterial occlusion(MCAO).Disturbance of energy metabolism after cerebral ischemia is considered to be the initiation point of cell damage after ischemia.After ischemia,the oxygen supply rapidly decreases,ATP is rapidly depleted,anaerobic glycolysis is strengthened,a large amount of lactic acid is generated,the respiratory function of mitochondria is inhibited,and the energy supply of the brain is rapidly reduced,which promotes intracellular Ca2+overload and reactive oxygen species(ROS)radical generation,leading to extensive brain injury.Therefore,it is of great significance to deeply explore the mechanism of energy metabolism disorder after cerebral ischemia and find effective therapeutic targetsTRIM31(Tripartite motif-containing protein 31)belongs to the TRIM family.It has E3 ubiquitin ligase activity and can specifically modify and degrade target proteins.It can participate in a variety of cell biological processes,including proliferation,differentiation,development,antiviral,canceration,and apoptosis However its role in cerebral ischemic injury has not been reported so far.It has been reported in the article that TRIM31 is associated with disorders of glucose metabolismTP53 induced glycolysis and apoptosis regulator(TIGAR)can reduce the activity of fructose 2,6 diphosphate Kinase,because its spatial structure is similar to the structure of 2,6 diphosphate fructose kinase,which is a key enzyme in the pentose phosphate pathway.So TIGAR can inhibit the glycolysis,reduce intracellular ROS levels and protect ischemic brain damage,but its regulatory mechanism is not yet clear.Therefore,this article will discuss the potential role of TRIM31 in focal cerebral ischemic injury and its molecular mechanismMethods1.Exploring the expression of TRIM3 1 in neurons after cerebral ischemia1.1 Establishing a Middle cerebral artery occlusion(MCAO)model in mice.Mice were decapitated at 6h,12h,24h,48h and 72h of MCAO.Changes in TRIM31 protein levels were detected by western blot.1.2 The expression and localization of TRIM31 in the ischemic penumbra region were detected by the immunofluorescence staining.1.3 Establishing a Oxygen glucose deprivation/Reperffusion(OGD/R)model in vivo.Primary neuronal cells were cultured.After 24 hours,the protein level of TRIM31 was detected by western blot.1.4 The expression of TRIM31 in primary neuronal cells was detected by the immunofluorescence staining2.Exploring the role of TRIM31 in cerebral ischemia2.1 The neurological function changes of TRIM31 knockout(TRIM31-/-)mice and Wide Type(WT)mice after cerebral ischemia were compared by Longa's neurological function score.2.2 The infarct volume of TRIM31-/-mice and WT mice after cerebral ischemia were compared by TTC staining.2.3 The cell morphology of TRIM31-/-mice and WT mice after cerebral ischemia were compared by HE staining3.Exploring the regulatory mechanism of TRIM31 on TIGAR3.1.The expression and localization of TIGAR in the ischemic penumbra region in C57 mice and TRIM31-/-mice were detected by the immunological staining.3.2 The TIGAR expression in C57 and TRIM31-/-mice were detected by western blot.3.3 Culturing PC 12 cells in vitro,transfecting with GFP-TRIM31 plasmid to over expressed TRIM31 and siTRIM31 to interfere TRIM31 expression.Changes in TIGAR protein levels were detected by western blot.4.Exploring the combination of TRIM31 and TIGAR4.1 The Co-localization of TIGAR and TRIM31 was detected by immunofluorescence staining in PC 12 cells.4.2 The endogenous combination of TRIM31 and TIGAR.were detected by using co-immunoprecipitation experiments performed with TRIM31 antibodies in PC 12 cells.4.3 The Exogenous combination of TRIM31 and TIGAR were detected by using co-immunoprecipitation experiments performed with FLAG antibodies in 293T cell which transfected with GFP-TRIM31 and MYC/FLAG-TIGAR.5.TRIM31 catalyzed the ubiquitination of TIGARHEK 293T cells were transfected with GFP-TRIM31,MYC/FLAG-TIGAR,and HA-Ubiquitin.Proteins were collected 24 hours later,and FLAG antibodies were used for co-immunoprecipitation experiments.The influence of TRIM31 on TIGAR ubiquitination was detected by western blot.6.Effect of interfering TRIM expression on glucose metabolism in PC 12 cells6.1 Cell survival rate were detected by CCK8 kit.6.2 Cellular ATP levels were detected by ATP kit.6.3 Cellular ROS levels were detected by Flow cytometry and immunofluorescence.6.4 The expression of G6PD in the PC 12 cells was detected by western blot.7.Effect of interfering TRIM expression on mitochondria in PC 12 cells7.1 Changes in mitochondrial membrane potential were detected by JC-1 kit7.2 The mtDNA deletion rate were detected by RT-pcr.8.Effects of TRIM31 knockout on glucose metabolism and mitochondrial homeostasis in miceChanges of G6PD,PGC-1a,MFN2,MFN1,and DRP1 protein levels in the ischemic penumbra were detected by western blot.9.Effects of knockdown of TIGAR expression on TRIM31-/-mice after cerebral ischemia9.1 Adeno-associated virus injection of AAV-shTIGAR in the brain of TRIM3 1-/-mice interferes with TIGAR expression9.2 The knockout efficiency of TIGAR was detected by western blot9.3 Comparison of neurological function after cerebral ischemia by Longa's neurological function score9.4 The neurological function changes of AAV-shTIGAR and AAV-GFP in TRIM31-/-mice after cerebral ischemia were compared by HE staining.9.5 G6PD protein levels were detected by western blot.Results1.In the WT mice after the treatment of MCAO,the protein level of TRIM31 is reduced in the penumbra of the mouse brain.Immunofluorescence staining showed that TRIM31 was distributed in mouse neurons,and the protein level of TRIM31 was significantly reduced after cerebral ischemia.It was verified in primary neuron culture in vitro that TRIM31 protein levels were reduced after the treatment of OGD/R2.Knocking out TRIM31 can improve neurological function after cerebral ischemia.TRIM31 knockout can reduce the neurological deficit score.TTC staining results showed that after MCAO,compared to the WT group,infarct volume was reduced in TRIM31-/-group.The HE staining results showed that knocking out TRIM31 can improve cell morphology to some extent.3.The immunofluorescence staining results showed that after MCAO,the increase protein level of TIGAR mainly occurred in the cortical neurons.TIGAR was increased up-regulated in the TRIM31 knock out mice.Western blot results further proved that after MCAO,TRIM31 can negatively regulate TIGAR expression,promoting the increasing of TIGAR protein level4.After interfering with the expression of TRIM31 in PC 12 cells,the protein level of TIGAR increased.In contrast,after over-expression of TRIM31,TIGAR protein levels decreased.5.The immunofluorescence staining showed that TRIM31 and TIGAR co-localized in PC 12 cells.The results of endogenous co-immunoprecipitation showed that in PC 12 cells,TRIM31 was able to bind to TIGAR,and the binding was reduced after the OGD/R stimulation.The results of exogenous co-immunoprecipitation also showed that TRIM31 was able to bind to TIGAR in 293T cells.6.Exogenous ubiquitination experiments found that in HEK 293T cells,by co-transfection of GFP-TRIM31 and MYC/FLAG-TIGAR,TRIM31 can promote ubiquitination of TIGAR7.After the treatment of OGD/R in PC 12 cells,interfering with the expression of TRIM31 can increase cell viability.Fluorescence,flow cytometry,and Western blot results showed that after OGD/R interfering with TRIM31 expression can reduce the increase of ROS and slow the decrease of G6PD.8.It was found in PC 12 cells that interference with TRIM31 expression can reduce mitochondrial damage after OGD/R.Mainly by slowing down the mitochondrial membrane potential decreasing,mitochondrial DNA deletion.9.In the MCAO model,compared to the WT group,the TRIM31-/-group can reduce the reduction of G6PD,slow down the mitochondrial synthesis-related proteins PGC-1a,mitochondrial fusion-related proteins MFN1,MFN2,and reverse the mitochondrial dynamics-related proteins DRP1 increasing10.Using adeno-associated virus to inject AAV-shTIGAR into the brain of TRIM31-/-mice to knock down the expression level TIGAR expression.Western blot results found that the knockdown efficiency was obvious.Fluorescence observation shows the spread range.By HE staining,neurological deficit score and western blot,it was found that knocking down TIGAR expression can abolish the protective effect of TRIM31 knockout after cerebral ischemia and reduce the increase of G6PDConclusionIn MCAO-induced cerebral ischemia model,TRIM31 can promote TIGAR ubiquitination degradation,thereby reduce pentose phosphate pathway,increase the production of ROS,aggravate mitochondrial damage,and mediate cerebral ischemic damage.