The Regulating Mechanism Of Ubiquitin-Like Protein FAT10 In HUVECs-mtROS By Stabilizing Mitochondrial Respiratory Chain Complex Protein NDUFAF1

Background:Oxidative stress is one of the main factors that damage the physiological function of vascular endothelial cells,and then cause vascular diseases such as hypertension and atherosclerosis..The pathophysiological mechanism of promoting oxidative stress in cardiovascular disease is not clear.As a member of the ubiquitin-like protein family,FAT10 has been found to play an important role in autophagy,apoptosis,inflammation,tumor,DNA damage response and so on.Mitochondrial reactive oxygen species(mtROS),an important participant in oxidative stress,are mainly produced by mitochondrial respiratory chain complexes I and III.The specific regulatory mechanism of FAT10 on mtROS has not been reported yet,and further research is needed.Objective:To explore the specific mechanism of ubiquitin-like protein FAT10 increasing mtROS by regulating the respiratory chain complexes.Method:Human umbilical vein endothelial cells(HUVECs)and HEK293 cells were used as research objects.HUVECs was conducted the following treatments:(1)Divided into 3 groups(n=6),which were overexpression negative virus control group,FAT10 overexpression group and FAT10 interference expression group,HUVECs was conducted the following two treatments: 1)using Mitochondrial Respiratory chain complex activity Detection Kit to detect the activity of Mitochondrial complex I and III,2)using Western-Blot to detect the expression of mitochondrial respiratory chain complex proteins;(2)using Western-Blot to detect the effect of cycloheximide(CHX),an inhibitor of protein synthesis,on the regulation of mitochondrial respiratory chain complex proteins expression by overexpressed FAT10(n=6);(3)treating HUVECs with MG132,and using Western-Blot to detect the degradation of mitochondrial respiratory chain complex proteins(n=6);(4)treating HUVECs with autophagy inhibitor CQ and autophagy agonist Rapamycin,and using Western-Blot to detect changes in mitochondrial respiratory chain complex proteins expression as autophagy levels decreased and increased(n=6);(5)using Western-Blot to detect the effect of overexpression of FAT10 on autophagy(n=4).HEK293 cells was conducted the following treatments: HEK293 cells were used to co-transfect the FAT10 plasmid and each mitochondrial respiratory chain complex protein plasmids,and a CO-IP experiments was performed to detect which mitochondrial respiratory chain complex protein interacts with FAT10;For the respiratory chain complex protein that interacts with FAT10,construct a mutant plasmid,and then perform CO-IP experiments with FAT10 to observe the change of interaction.Result:(1)Compared with the ad-Ctrl group,the activity of mitochondrial respiratory chain complex I was increased in the ad-Fat10 group(p <0.01),the activity of mitochondrial respiratory chain complex I was decreased in the sh-Fat10 group(p <0.05);Compared with the ad-Fat10 group,the activity of mitochondrial respiratory chain complex I in the sh-Fat10 group was reduced(p <0.05);there was no significant difference in the activity of mitochondrial respiratory chain complex III in each group;The results showed that FAT10 could regulate the activity of mitochondrial respiratory chain complex I;(2)Compared with the ad-Ctrl group,the expression of mitochondrial respiratory chain complex proteins NDUFS2,NDUFAF1 and NDUFC2 were significantly increased in ad-Fat10 group(p <0.01),and the expression levels of Cytochrome C,COX ?,and SDHA proteins were not significantly changed in ad-Fat10 group;Compared with the ad-Ctrl group,the expression of mitochondrial respiratory chain complex proteins NDUFS2,NDUFAF1 and NDUFC2 were decreased in sh-Fat10 group(p <0.05),and the expression levels of Cytochrome C,COX ?,and SDHA proteins were not significantly changed in sh-Fat10 group;Compared with the ad-Fat10 group,the expression levels of mitochondrial respiratory chain complex proteins NDUFS2,NDUFAF1 and NDUFC2 were decreased in the sh-Fat10 group(p <0.05,p <0.01,p <0.05),and the expression of Cytochrome C,COX ?,and SDHA proteins were not significantly changed significantly in the sh-Fat10 group;The results showed that FAT10 could regulate the expression of respiratory chain complex proteins.(3)CO-IP experiments showed that FAT10 interacted with mitochondrial respiratory chain complex protein NDUFAF1,the results showed that FAT10 may directly regulate the expression of respiratory chain complex protein NDUFAF1.(4)Using cycloheximide to treat HUVECs transfected into ad-Fat10 group and ad-Ctrl group,NDUFAF1 protein increased in ad-Fat10 group(p <0.05);treating HUVECs with MG132,the expression of endogenous NDUFAF1 protein expression had no significant change with the prolongation of MG132 treatment time;the treatment of HUVECs with autophagy inhibitor CQ,the expression of NDUFAF1 protein increased with the decrease of autophagy level(p <0.05);the treatment of HUVECs with autophagy agonist Rapamycin,as the level of autophagy increased,the expression of NDUFAF1 protein decreased(p <0.001);The results showed that NDUFAF1 protein was regulated by autophagy.(5)Compared with the ad-Ctrl group,the level of autophagy in the ad-Fat10 group was decreased,indicating that overexpression of FAT10 can reduce the level of autophagy(p <0.01).Conclusion:(1)Overexpression of FAT10 increased the activity of mitochondrial respiratory chain complex I and the expression of mitochondrial respiratory chain complex proteins NDUFS2,NDUFAF1 and NDUFC2.(2)FAT10 interacts directly with the mitochondrial respiratory chain complex protein NDUFAF1.(3)NDUFAF1 protein is regulated by autophagy.(4)Overexpression of FAT10 decreases the level of autophagy.