UBL4A-like Ubiquitination At ALDH1L1-812K Site Induces SEC62-dependent Reticulophagy And Mediates Apelin-13/APJ Promoting Monocyte-vascular Endothelial Cells Adhesion

Apelin,as a vasoactive peptide,is an endogenous ligand of APJ receptor.Studies have showed that Apelin/APJ is mainly distributed in the cardiovascular system,especially significantly expressed in vascular smooth muscle cells,cardiomyocytes and vascular endothelial cells,which suggests that Apelin/APJ system is closely related to the physiological and pathological regulation of the cardiovascular system.Atherosclerosis?AS?is the pathological basis of cardiovascular diseases.Monocyte-vascular endothelial cells adhesion is an important link in the development of AS.Our previous research revealed that Apelin-13 can promote reactive oxygen species?ROS?to induce monocyte-vascular endothelial cells adhesion,but the specific mechanism has not yet been elucidated.Therefore,further in-depth study of the specific mechanism of Apelin/APJ system to promote monocyte-vascular endothelial cells adhesion will have important scientific significance and clinical value for the prevention and treatment of AS.According to literature,massive intracellular ROS production can induce endoplasmic reticulum stress,which finally can promote the production of reticulophagy.Under normal physiological conditions,intracellular reticulophagy can occur at a low level,which helps to clear the damaged endoplasmic reticulum membrane and maintain the normal shape and function of the endoplasmic reticulum.When cells are stimulated,the level of reticulophagy will increase significantly,resulting in a series of pathological changes in the body.Therefore,exploring the specific regulation process of reticulophagy induced by Apelin/APJ system will be the key issue of this research.The important basis of reticulophagy is the activation of reticulophagy receptor protein.Reports has confirmed that SEC62 is a type of reticulophagy receptor protein which can participate in the regulation of endoplasmic reticulum stress.By using bioinformatics,we found that there may be an interaction between Apelin and SEC62,which suggests that Apelin may activate SEC62 to induce reticulophagy.Further review of the literature,the occurrence of endoplasmic reticulum stress in cells is related to folate metabolism disorders,and ALDH1L1 is an important enzyme involved in folate metabolism,thus we speculated that Apelin may promote the occurrence of SEC62-dependent reticulophagy by inducing the expression of ALDH1L1.However,how ALDH1L1 in the cytoplasm affects the process of reticulophagy is still unclear and further investigation is needed.ALDH1L1 is a member of the tail-anchored protein family,and its C-terminus can be anchored to the plasma membrane to perform biological functions.Ubiquitin-like modification is a new type of protein post-translational modification process,which can transport modified protein and locate on the plasma membrane to exert biological effects.UBL4A is a small molecule ubiquitin-like protein.It is reported in the literature that it can perform ubiquitin-like modification on tail-anchored proteins.Using bioinformatics software found that the 812 lysine of the tail anchored protein ALDH1L1 may be an important site for ubiquitin-like modification.In addition,anchoring of the tail anchor protein ALDH1L1 to the plasma membrane also requires the assistance of the tail anchor protein recognition factor ASNA1.Based on these findings,our project proposes the scientific hypothesis that UBL4A-like ubiquitination at ALDH1L1-812K site induces SEC62-dependent reticulophagy and mediates Apelin-13/APJ promoting monocyte-vascular endothelial cells adhesion.To verify this hypothesis,the research content will be divided into three parts:?1?SEC62-dependent reticulophagy mediated Apelin-13/APJ promoting monocyte-vascular endothelial cells adhesion;?2?Tail anchoring Protein ALDH1L1 induces SEC62-dependent reticulophagy and mediates Apelin-13/APJ promoting monocyte-vascular endothelial cells adhesion;?3?UBL4A-like ubiquitination at ALDH1L1-812K site induces SEC62-dependent reticulophagy mediates Apelin-13/APJ to promote monocyte-vascular endothelial cells adhesion.In this study,we firstly linked the Apelin/APJ system,ubiquitin-like modification tail-anchored protein ALDH1L1,SEC62-dependent reticulophagy and monocyte-vascular endothelial cells adhesion,which try to clarify that Apelin-13induces UBL4A-like ubiquitination ALDH1L1 at the site of 812K,and modified ALDH1L1 is anchored to the endoplasmic reticulum membrane under the action of ASNA1,which finally induce SEC62-dependent reticulophagy.These are important academic innovations that Apelin/APJ system can promote the adhesion of monocyte-vascular endothelial cells,which provide an important experimental basis for further revealing the prevention and treatment of AS with APJ drugs as targets.Part? SEC62-dependent reticulophagy mediated Apelin-13/APJ promote monocyte-vascular endothelial cells adhesionObjective:Feed ApoE gene knockout(apopoprotein E deficiency,ApoE^-/-)mice with high fat and cholesterol to construct an atherosclerosis model,exploring the effects of Apelin-13 and APJ antagonist F13A on the plaques of aortic sinus in atherosclerotic mice;3 pairs of paraffin wax blocks for autopsy coronary arteries were gifted from Sun Yat-sen University to explore the expression of cells adhesion molecules ICAM1,VCAM1 and reticulophagy receptor protein SEC62;vitro experiments to investigate whether Apelin-13 promotes monocyte-vascular endothelial cells adhesion by inducing SEC62-dependent reticulophagy.Methods:A total of 24 male 8-week-old ApoE^-/-mice were randomly divided into ApoE^-/-group,ApoE^-/-+Apelin-13 group,ApoE^-/-+Apelin-13+F13A group,each group had eight mice were fed with high-fat cholesterol diet,and detected the formation of aortic sinus plaque in ApoE^-/-mice after 8 weeks.Subsequently,the above groups were injected intraperitoneally with saline,Apelin-13,Apelin-13+F13A once a day for 28 consecutive days.The injection volume of Apelin-13and F13A were 5 mg/kg per day.After 28 days,the mice were sacrificed,the aortic arch of the mice was isolated,paraffin sections of aortic sinuses were made,and the size of AS plaques at the aortic sinuses was detected by HE staining.Immunohistochemistry was used to detect the expression of ICAM1,VCAM1 and SEC62 in aortic plaque.In human autopsy specimens,HE staining was used to observe the size of plaque in coronary arteries,and immunohistochemistry was used to detect the expression of ICAM1,VCAM1,BIP,CHOP,and SEC62 in the coronary arteries.Pretreatment with F13A,endoplasmic reticulum stress inhibitor Salubrinal,autophagy inhibitor 3MA,Western blot,immunofluorescence and transmission electron microscopy were used to detect the above factors on Apelin-13-induced the expression of ICAM1,VCAM1,SEC62,BECLIN1,LC3,the formation of endoplasmic reticulum autophagosomes and the effect of monocyte-vascular endothelial cells adhesion.Results:HE staining results showed that F13A could inhibit Apelin-13-induced the increase size of aortic sinus atherosclerotic plaque in ApoE^-/-mice;immunohistochemical results showed that F13A inhibited Apelin-13-induced the expression of ICAM1 in plaques of ApoE^-/-mice;HE staining results showed that obvious plaques can be seen in coronary arteries of patients with atherosclerosis;immunohistochemistry results showed the increased expression of ICAM1,VCAM1,BIP,CHOP,SEC62,BECLIN1,LC3 in coronary arteries of autopsy atherosclerosis patients compared with the control group.The above in vitro experimental results indicated that Apelin-13can promote the development of AS,which may be related to the activation of reticulophagy.Further research found that Apelin-13 can promote the expression of reticulophagy-related proteins SEC62,BECLIN1,LC3 in vascular endothelial cells in dose?0¹.0umol/L?and time?0²4h?dependent;transmission electron microscope observed that F13A can inhibit the formation of endoplasmic reticulum autophagosomes in vascular endothelial cells induced by Apelin-13;SEC62 siRNA inhibited the expression of ICAM1 and VCAM1 in vascular endothelial cells induced by Apelin-13 and the adhesion ofmonocyte-vascular endothelial cells,all these results indicated that SEC62-dependent reticulophagy may be involved in Apelin-13 promoting monocyte-vascular endothelial cells adhesion.To further clarify the above mechanism,pretreatment endoplasmic reticulum stress inhibitor Salubrinal and autophagy inhibitor 3MA in vascular endothelial cells,Western blot and immunofluorescence showed that the above treatments can attenuate the expression of SEC62,ICAM1,VCAM1,decrease the co-localization of LC3 and SEC62 in vascular endothelial cells induced by Apelin-13,and inhibit the formation of endoplasmic reticulum autophagosomes.Conclusions:?1?Apelin-13 can promote the increase of AS plaque in the aortic sinus of ApoE^-/-mice,APJ antagonist F13A can inhibit the increased size of aortic plaque induced by Apelin-13 in ApoE^-/-mice.All results showed that Apelin/APJ system may be an important target for prevention and treatment of AS.?2?Apelin-13 can promote the expression of reticulophagy protein SEC62,which induce reticulophagy to promote monocyte-vascular endothelial cells adhesion.Part?Tail-anchored protein ALDH1L1 induces SEC62-dependent reticulophagy mediates Apelin-13/APJ promoting monocyte-vascular endothelial cells adhesionObjective:To investigate the effect of Apelin-13 on the expression of the tail-anchored protein ALDH1L1,and whether activated ALDH1L1can induce SEC62-dependent reticulophagy to promote monocyte-vascular endothelial cells adhesion.Methods:Immunohistochemistry was used to observe the expression of ALDH1L1 in the aortic AS plaques of ApoE^-/-roup,ApoE^-/-+Apelin-13group,ApoE^-/-+Apelin-13+F13Agroup;immunhistochemistry was used to detect the expression of ALDH1L1 in AS plaques of coronary arteries.Treat vascular endothelial cells with different concentrations?0.001,0.01,0.1,1umol/L?of Apelin-13 for 24hours or use 1 umol/L Apelin-13 for different times?0h,3h,6h,12h,24h?,Western blot was used to detect the dose and time effect of ALDH1L1 induced by Apelin-13.Using ALDH1L1 siRNA,Western blot was used to detect the effects of SEC62,ICAM1,and VCAM1 on vascular endothelial cells induced by Apelin-13,and immunofluorescence was used to observe the colocalization of SEC62 and LC3,and endoplasmic reticulum protein KDEL and LC3.All these treatments try to further confirmed whether Apelin-13 can affect the expression of SEC62through the regulation of ALDH1L1,which finally affects the regulation of reticulophagy.In addition,construction of ALDH1L1 high expression plasmid,Western blot was used to detect the expression of SEC62 in vascular endothelial cells.Results:Immunohistochemical results showed that F13A inhibitedthe expression of ALDH1L1 in plaques aortic of ApoE^-/-mice treatment with Apelin-13;autopsy immunohistochemical results showed that the expression of ALDH1L1 in coronary plaques of patients with atherosclerosis was significantly increased than that of controls group.The above results indicated that the increase expression of ALDH1L1may be proportional to the increase in plaque area.The results of vitro experiments showed that Apelin-13 increased the expression of ALDH1L1 in vascular endothelial cells in a concentration-dependent?0¹.0?M?and time?0²4h?-dependent manner,F13A inhibited the expression of ALDH1L1 in vascular endothelial cells induced by Apelin-13.Immunofluorescence results showed that ALDH1L1 siRNA attenuated the colocalization of SEC62 and LC3,endoplasmic reticulum proteins KDEL and LC3 in vascular endothelial cells induced by Apelin-13.These suggest that ALDH1L1 may be an upstream protein can activate SEC62,and ALDH1L1 siRNA can attenuate the occurrence of SEC62-dependent reticulophagy induced by Apelin-13.Western blot results also showed that high expression of ALDH1L1 can increase the expression of SEC62 in endothelial cells,which further confirmed that the increased expression of SEC62 depends on the activation of ALDH1L1.Conclusions:?1?Apelin-13 can upregulate the expression of ALDH1L1 in vascular endothelial cells,which induce SEC62-dependent reticulophagy and promote monocyte-vascular endothelial cells adhesion.Part?UBL4A-like ubiquitination ALDH1L1-812K site mediates Apelin-13/APJ to promote monocyte-vascular endothelial cells adhesionObjective:UBL4A is a ubiquitin-like protein that can ubiquitin-like modification of substrate proteins.Bioinformatics predicted that the important ubiquitin-like modification site of ALDH1L1 is the 812 lysine site.Literature showed that the tail-anchored protein recognition factor ASNA1 can transport tail-anchored proteins from the cytoplasm to the endoplasmic reticulum.Therefore,this section focuses on whether Apelin-13 can modify the 812 lysine site of ALDH1L1 through UBL4A ubiquitination to promote monocyte-vascular endothelial cells adhesion.Methods:Immunohistochemistry was used to detect the expression of UBL4A and ASNA1 in aortic plaque of ApoE^-/-mice and coronary plaque in autopsy patients with atherosclerosis.Western blot further examined the expression of UBL4A and ASNA1 in dose-effect and time-effect induced by Apelin-13 and F13A in vascular endothelial cells.It is speculated that UBL4A is the upstream regulatory protein of ALDH1L1.To verify this idea,Western blot and immunofluorescence was used to detect the effect of UBL4A siRNA on the expression of ALDH1L1,SEC62,LC3,ICAM1,VCAM1 in vascular endothelial cellsand monocyte-vascular endothelial cells adhesion,transmission electron microscopy was used to detect the effect of UBL4A siRNA on formation of endoplasmic reticulum autophagosome.To further verify whether UBL4A can ubiquitin-like modification of ALDH1L1 at the site of 812lysine,mutation of lysine at position 812 to arginine,using ALDH1L1-812K mutant plasmids,Western blot and immunofluorescence was used to observe the expression of SEC62,ICAM1,VCAM1 and monocyte-vascular endothelial cells adhesion.In addition,Western blot also detected the effect of ASNA1 siRNA on the expression of BIP,SEC62,ICAM1,and VCAM1 in vascular endothelial cells induced by Apelin-13.Results:Immunohistochemical results showed that F13A inhibited the increase expression of UBL4A and ASNA1 in the aorta of ApoE^-/-mice induced by Apelin-13;autopsy immunohistochemical results showed that the expression of UBL4A and ASNA1 in coronary artery of patients with atherosclerosis was higher than control group.All results suggested that the increased expression of UBL4A and ASNA1 may be positively correlated with the size of atherosclerotic plaque.The results of vitro experiments further confirmed that Apelin-13 promoted the increased expression of UBL4A and ASNA1 in a concentration-dependent?0¹.0?M?and time?0²4h?dependence,and F13A inhibited the expression of UBL4A and ASNA1 in vascular endothelial cells induced by Apelin-13.Western blot and immunofluorescence results showed that UBL4A siRNA inhibited the expression of ALDH1L1,SEC62,LC3,ICAM1,and VCAM1 in vascular endothelial cells induced by Apelin-13,reduced the formation of endoplasmic reticulum autophagosomes,and inhibited monocyte-vascular endothelial cells adhesion.Mutation of the 812 lysine site of ALDH1L1,Western blot and immunofluorescence observations revealed that the expression of SEC62,ICAM1,and VCAM1 in vascular endothelial cells was decreased,and monocyte-vascular endothelial cell adhesion was inhibited.In addition,Western blot also found that ASNA1 siRNA can inhibit the expression of BIP,SEC62,ICAM1,and VCAM1 in vascular endothelial cells induced by Apelin-13.Conclusions:Apelin-13 can up-regulate the expression of UBL4A,which ubiquitin-like modification of ALDH1L1 at the site of 812 lysine.with the assistance of ASNA1,Ubiquitin-like modified ALDH1L1 can anchor to the endoplasmic reticulum membrane and activate SEC62-dependent reticulophagy,finally promoting monocyte-vascular endothelial cells adhesion.