UBL3 Ubiquitinates MAPK15 And Induces The Phosphorylation Of PEX5 Enhancing Pexophagy That Mediates Apelin-13/APJ Promoting The Proliferation Of Vascular Smooth Muscle Cells

Apelin/APJ system is involved in a variety of biofunctional processes,including the vascular growth,internal environment homeostasis,energy metabolism and other physiological processes.As a vasoactive peptide,apelin is an endogenous ligand of APJ receptor,is closely related to the physiological and pathological regulation of the cardiovascular system.Atherosclerosis（AS）is one of the most important pathological bases of cardiovascular system diseases and peripheral vascular diseases.Vascular smooth muscle cells（VSMCs）abnormalities exacerbate the process of AS such as DNA damage,mitochondrial dysfunction and autophagy.Our previous study has revealed that apelin-13 can promote reactive oxygen species（ROS）generation to induce the proliferation of vascular smooth muscle cells.Therefore,further study is needed to be clarified the exact mechanism of apelin/APJ system promoting the proliferation of vascular smooth muscle cells,which may be great significance for the prevention and treatment of AS.In the physiological condition,the intrcellular pexophagy is stable at a low level,which can be helpful to remove damaged peroxisomes,and maintain the level of oxidative metabolism in cells.However,pexophagy will be eventually out-of-balance when cells are cells are stimulated by oxidative stress,hypoxia and other external stimulation.The pexophagy will be excessive activation,leading a series of pathological changes in the body.It is reported that Peroxisomal biogenesis factor 5（PEX5）is the key molecule receptor of pexophagy,which is involved in the regulation of the pexophagy.Bioinformatic analysis revealed that there might be protein-protein interactions between apelin and PEX5.After reviewing literature,PEX5 can be phosphorylated by some phosphoprotein kinases,and promote the pexophagy.Therefore,apelin-13 might promote the phosphorylation modification of PEX5 to induce the pexophagy.The specific mechanism of the phosphorylation modification of PEX5 is unclear and needed to be further explored.Mitogen-activated protein kinases 15（MAPK15）is a phosphorylated kinase that is tightly controlled by the protein ubiquitin protein system.MAPK15 might be involved in the phosphorylation modification of PEX5,and the phosphorylation site was speculated to be 589 serine by using bioinformatics analysis.However,the specific mechanism and cellular pathways of pexophagy is also unclear,which is induced by MAPK15 phosphorylated PEX5.Thus,it is extremely important to identify the key regulatory mechanism of apelin-13 on promoting pexophagy induced by PEX5 phosphorylation via MAPK15 induced.The ubiquitination of protein is a process of protein translational modifications,which can covalently modify individual amino acid residues,and can change their physicochemical properties and conformation,and then affect the function of protein.Ubiquitin-like protein 3（UBL3）is a small ubiquitinated protein.Consulting document reported that MAPKs can be fused with UBL3.By using bioinformatics analysis,the prediction,indicated that the lysine 42 site of MAPK15 may be an important site for ubiquitin-like modification.Meanwhile,UBL3may ubiquitin-like-modificate MAPK15.According to those finding,the scientific hypothesis is UBL3ubiquitinates MAPK15 induces the phosphorylation of PEX5 enhance pexophagy and mediates apelin-13/APJ promoting the proliferation of vascular smooth muscle cells.To verify this hypothesis,this research is divided into three parts:1^stapelin-13 induce pexophagy in VSMCs,2^ndapelin-13/APJ system activates MAPK15 ubiquitination through UBL3,and enhances pexophagy via the phosphorylation of PEX5,3^rdUBL3might ubiquitinate MAPK15 induces the phosphorylation of PEX5,promotes pexophagy and mediates apelin-13/APJ promoting the proliferation of Vascular smooth muscle cells.The above research has made an important academic innovation for the proliferation of vascular smooth muscle cells via apelin/APJ system and provides a new theoretical basis for the further study of AS with APJ drugs as targets.Part I Apelin-13/APJ induce pexophagyObjective:To explore the effects of apelin-13 on the number of peroxisomes.To observe the expression of pexophagy relative proteins PEX5 and LC3 in the vascular smooth muscle cells of Apo E^-/-atherosclerotic mice and coronary arteries.To investigate whether apelin-13 promotes pexophagy in vascular smooth muscle cells.Methods:Electron microscopy,Immunoelectron microscopy and Immunohistochemistry were used to detect the effects of apelin-13on the number of peroxisomes and the expression of pexophagy relative proteins PEX5 in the vascular smooth muscle cells.Immunohistochemistry was used to detect the expression of pexophagy relative proteins PEX5 in Apo E^-/-atherosclerotic mice and atherosclerotic coronary arteries.Detecting the F13A and autophagy inhibitor Bafilomycin A1 on the effects of apelin-13 induced the expression of PEX5 and LC3 by using immunofluorescence and western blot.Western blot was used to detect the effects of PEX5 si RNA on the expression of PEX5 and LC3 in HA-VSMCs.Immunofluorescence was used to detect the effects of PEX5 si RNA on the expression and co-localization of PEX5and LC3 in HA-VSMCs.Western blot was used to detect the effects of autophagy inhibitor Bafilomycin A1 on the expression of LC3 in HA-VSMCs.Results:Apelin-13 decreased the number of peroxisomes and induced pexophagy.Meanwhile,the number of peroxisomes and fluorescence intensity were decreased in vascular smooth muscle cells of Apo E^-/-mice.Compared with normal coronary arteries,PEX5 was increased in vascular smooth muscle cells of AS coronary arteries,and the expression of pexophagy relative proteins PEX5 and LC3 were increased in vascular smooth muscle cells of Apo E^-/-atherosclerotic mice.apelin-13 induced the co-localization of PEX5 and LC3 in HA-VSMCs.The above results showed that apelin-13 promoted the process of AS,and the reason might be relative with the activation of pexophagy.Further results in vitro found that apelin-13 can promote the expression of pexophagy-related proteins PEX5,LC3 in vascular smooth muscle cells in dose and time dependent,and F13A can decreased the effect of apelin-13 promote the expression of pexophagy-related proteins PEX5and LC3 in vascular smooth muscle cells.Western blot showed that autophagy inhibitor Bafilomycin A1 inhibit the co-localization of PEX5and LC3,that induced by apelin-13,in HA-VSMCs by using immunofluorescence.PEX5 si RNA decreased the expression of LC3 in HA-VSMCs and inhibit the co-localization of PEX5 and LC3.Over expression PEX5 can promote the expression of PEX5 and LC3 in HA-VSMCs,and Bafilomycin A1 inhibit the expression of LC3 induced by apelin-13.Summary:Apelin-13 can up-regulate the expression of pexophagy relative proteins PEX5,and which induce pexophagy.PartⅡApelin-13 induce UBL3 ubiquitinates MAPK15 induces the phosphorylation of PEX5 enhance pexophagyObjective:To investigate the effect of apelin-13 on the expression of UBL3 MAPK15 and PEX5,and their relationship between them.Methods:Firstly,the relationship between MAPK15,PEX5 and apelin-13 were predicted by using bioinformatics analysis,and the phosphorylated modification site was predicted that MAPK15 can phosphorylate PEX5.Co-Immunoprecipitation was used to find out whether was an interaction between PEX5 and MAPK15,and the phosphorylation sites of PEX5 were analyzed by mass spectrometry.Immunohistochemistry was used to detect the expression of MAPK15 of vascular smooth muscle cells in Apo E^-/-atherosclerotic mice and atherosclerotic coronary arteries.Western blot and Immunofluorescence were used to detect the effect induced by apelin-13,F13A,MAPK15-si RNA,pc DNA-PEX5^Mut589S,pc DNA-MAPK15,and Bafilomycin A on expression and co-localization of PEX5 and LC3 in HA-VSMCs.Moreover,the relationship between UBL3,MAPK15,PEX5 and apelin-13 were predicted by using bioinformatics analysis,and the site was predicted that UBL3 can be ubiquitin-like modificated by MAPK15.Co-Immunoprecipitation was used to find out whether was an interaction between MAPK15 and UBL3.Immunohistochemistry was used to detect the expression of UBL3 of vascular smooth muscle cells in Apo E^-/-atherosclerotic mice and coronary arteries.Western blot and Immunofluorescence were used to detect the effect induced by apelin-13,F13A,UBL3-si RNA,pc DNA-MAPK15^Mut42Kon expression and co-localization of PEX5 and LC3 in HA-VSMCs.Results:The results of bioinformatics analysis,co-Immunoprecipi tation and mass spectrometry suggested that there was an interaction between UBL3,MAPK15 and PEX5.MAPK15 can phosphorylate modification of PEX5 at the site of 589 serine.UBL3 can be ubiquitin-like modification of MAPK15 at the site of 42 lysine.The expression of MAPK15 and UBL3 of Apo E^-/-atherosclerotic mice and autopsy coronary arteries were increased,and apelin-13 could induce the expression of MAPK15 of vascular smooth muscle cells in Apo E^-/-atherosclerotic mice.Results in vitro found that apelin-13 can promote the expression of pexophagy-related proteins MAPK15 and UBL3 in dose and time dependent,and F13A can decreased the effect of apelin-13promote the expression of MAPK15 in HA-VSMCs.MAPK15 si RNA,pc DNA-PEX5^Mut589S,UBL3-si RNA,pc DNA-MAPK15^Mut42Kdecreased the expression of PEX5 and LC3 that induced by apelin-13 and Rapamycin in HA-VSMCs.MAPK15 si RNA,pc DNA-PEX5^Mut589S,pc DNA-MAPK15^Mut42Kdecreased the co-localization of PEX5 and LC3that induced by apelin-13 in HA-VSMCsSummary:（1）Apelin-13 induced the phosphorylation modified PEX5 at 589S site by MAPK15 and promoted pexophagy;（2）Apelin-13induced the ubiquitin-like modified MAPK15 at 42K site by UBL3 and promoted pexophagy.PartⅢUBL3 ubiquitinates MAPK15 induces the phosphorylation of PEX5 enhance pexophagy and mediates apelin-13/APJ promoting the proliferation of Vascular smooth muscle cellsObjective:To investigate the effect of apelin-13 on the expression of UBL3 and the ubiquitination of MAPK15,and activated MAPK15 can induce the phosphorylation of PEX5,which promote pexophagy,eventually promote the proliferation of vascular smooth muscle cells.Methods:Immunohistochemistry was used to detect the expression of PCNA of vascular smooth muscle cells in Apo E^-/-atherosclerotic mice and atherosclerotic coronary arteries.The technique of CCK8 and Brdu were used to detect the effect of apelin-13 on the proliferation of HA-VSMCs.Western blot was used to detect the dose and time effect of PCNA induced by apelin-13 in HA-VSMCs and detecting the F13A on the effects of apelin-13 induced the expression of PCNA.The technique of CCK8,Brdu and Western blot were used to detect the effect of PEX5si RNA,MAPK15 si RNA,UBL3 si RNA on the expression of PCNA and the proliferation of HA-VSMCs.Immunohistochemistry was used to detect the effect of PEX5-si RNA and pc DNA-PEX5 on the expression of PCNA.Lastly,the technique of CCK8,Brdu and western blot were used to detect the effect of pc DNA-PEX5^Mut589S,pc DNA-MAPK15^Mut42Kon the proliferation of HA-VSMCs and the expression of PCNA.Results:The expression of PCNA of Apo E^-/-atherosclerotic mice and atherosclerotic coronary arteries were increased,and apelin-13 could induce the expression of PCNA of vascular smooth muscle cells in Apo E^-/-atherosclerotic mice.Results in vitro found that apelin-13 can promote the expression of proliferation-related proteins PCNA in vascular smooth muscle cells in dose and time dependent,and F13A can decreased the effect of apelin-13 promote the expression of PCNA in vascular smooth muscle cells.PEX5 si RNA、MAPK15 si RNA and UBL3 si RNA decreased the expression of PCNA induced by apelin-13 in HA-VSMCs,and also inhibited the proliferation of vascular smooth muscle cells.Bafilomycin A1 decreased the expression of PCNA induced by overexpression PEX5.pc DNA-PEX5^Mut589Sand pc DNA-MAPK15^Mut42Kdecreased the expression of PCNA induced by apelin-13 and Rapamycin in vascular smooth muscle cells.Summary:Apelin-13 can promote the ubiquitination of MAPK15at 42K site by UBL3,and induced the phosphorylation of PEX5 at 589S site,which promote pexophagy and eventually promote the proliferation of vascular smooth muscle cells.Conclusions:（1）Apelin-13 induces PEX5-dependent pexophagy,（2）Apelin-13 activates UBL3 ubiquitination of MAPK15,and the ubiquitination site is 42K,that mediated pexophagy induced by apelin-13,（3）MAPK15 phosphorylates PEX5 at the site of 589S,and mediated pexophagy induced by apelin-13,（4）Apelin-13 promotes the proliferation of HA-VSMCs through the UBL3-MAPK15-PEX5-pexophagy pathway.