Involvment Of XIAP-regulated COMMD1 Protein Degradation In Lead Exposure-induced Aberrance Of Copper Transport In Brain Barriers

Lead is one kind of heavy metal contaminant which widely exists in nature.A great number of studies have proved that lead exposure has obvious toxic effects on central nervous system.Lead exposure can cause damages on advanced nervous functions during developmental period,e.g.cognitive disorder,distraction and abnormal behaviors.Recent studies have found out that lead exposure also has a connection to neurodegenerative diseases,including but not only Alzheimer's Disease(AD)and Parkinson's Disease(PD).The mechanism of the neurotoxicity of lead has not been completely revealed yet.Existing researches showed that lead exposure could affect immediate early gene expression,interfere with the expression and functions of neurotransmitter and its receptor,damage synaptic plasticity,induce abnormal activation of glial cells,destroy the structure and function of brain barrier,disrupt metabolism of trace elements in brain,etc.All the facts above suggest that the mechanism of lead neurotoxicity is a complicated progress which is related to gene expression and microenvironment in brain.Copper is one of the essential trace elements in the body.Cooper content in brain is second only to liver in one's body.Appropriate copper level plays a vital role in the development and the function maintenance of brain.But on the other hand,free cooper ion is a strong oxidant.With too high level of cooper ion,the balance of oxidation-reduction in brain may be destroyed,and this may have a close connection to the etiology of some neurodegenerative diseases.Intracellular copper metabolism relies on interaction of various copper transports and molecular chaperones.Copper ion enters the cells through copper transporter 1(CTR1),and then be transported to mitochondria and other organelles with the help of molecular chaperones(COX17,ATOX1 etc.).The excretion of the intracellular copper ions mainly depends on the transportation by ATP7 A or ATP7 B located in Golgi network.Some studies pointed out that Copper Metabolism MURR1Domain-containing 1(COMMD1)can regulate cells' copper metabolism by affecting ATP7 A and ATP7 B.It has been confirmed that XIAP is the key to regulate COMMD1 protein level,which can mediate COMMD1 ubiquitin and promote its degradation.Our past research has noticed that lead exposure can affect the expression of copper transporter like CTR1?ATP7A,resulting in copper accumulation in cells.This indicates that lead may cause oxidative stress injury by disrupting copper metabolism in brain.Nevertheless,there is no relative report about whether XIAP and COMMD1 participates in the copper metabolism in brain under the lead exposure,and the details of its molecular mechanism are not reported yet.Brain barrier,mainly including blood brain barrier(BBB)and blood-cerebrospinal fluid barrier(BCB),is an important structure to maintain the balance of the substance in brain.In this research,we establish a cell model of BBB and BCB in vitro to study the effect of lead on the copper ion transport in brain barrier cells,investigate the role of COMMD1 in the copper metabolism disorder under lead exposure,and try to find out the key molecules in the upstream and downstream.This will help to clarify the key part of copper ions disorder in the brain caused by lead,and lay a solid foundation for further study of molecular mechanism.Objective:In this research,we take cerebral microvascular endothelial cell strain RBE4 and choroid plexus epithelial cell strain Z310 from rats as experiment subjects,and establish lead exposure models respectively of BBB and BCB in vitro.The aim is to investigate lead's effect on cellular copper metabolism and clarify whether XIAP and COMMD1 plays a role in lead's effect on copper transporters(e.g.ATP7 B and ATP7 A etc.).At the same time,we expect to figure out the process of intracellular copper accumulation induced by lead and discuss molecular mechanism in the upstream and downstream.Methods:1.Culture RBE4 and Z310 in vitro,and establish models of BBB and BCB under lead exposure separately.Use MTT to select the appropriate dose of lead exposure.2.Analyze intracellular copper level by atomic absorption spectrophotometry(AAS).3.Use PR-PCR to analyze m RNA's expression level of XIAP,COMMD1,ATP7 B and ATP7 A in cells after being exposed to lead;Use Western Blot to detect protein's expression level of XIAP,COMMD1,ATP7 B and ATP7 Ain cells after being exposed to lead.4.Use si RNA interference technology to inhibit the expression of COMMD1,and observe the expression level of ATP7 A and ATP7 B as well as changes in cell's absorption amount of copper.5.Construct the plasmid which highly expresses XIAP,and observe the changes of the expression level of COMMD1,ATP7 A,ATP7B and cell's absorption amount of copper.6.Construct cells which are transfected by different domain segments of COMMD1,and observe the changes of ATP7 B expression.Clarify which part of COMMD1 is the functional domain to regulate ATP7B's expression.(This is not presented in the result)7.Design two kinds of plasmids,the wild type and the one with mutant XIAP segment in E3 ubiquitin ligase.Use the two kinds of plasmids to transfect cells separately,and observe the changes in COMMD1's expression.Clarify which part of XIAP is the functional domain to regulate COMMD1's expression.Results:1.Lead exposure's effect on copper levels of REB4 and Z310 cells.REB4 and Z310 cells were divided in 3 groups,and separately cultured with 2.5,5,10?mol/L lead acetate for 24 hours.Then each group was incubated in the incubator with Cu Cl2 of 5?mol/L at 37?.The result of AAS shows that compared to the control group,the copper level in both cells under lead exposure is significantly increased(p<0.05)with dose-effect relationship.2.Lead exposure's effect on m RNA and protein expression of XIAP,COMMD1,ATP7 B and ATP7 A in REB4 and Z310 cells.According to Western Blot results,compared to the control group,after 24 hours in2.5,5,10?mol/L lead acetate or in 10?mol/L lead acetate for 24 and 48 hours,both kinds of cells have a significant decrease in XIAP,ATP7 B and ATP7 A protein,while the COMMD1 protein has a significant increase(p<0.05),and there were dose-effect and time-effect relationships.However,compared to the control group,m RNA's level of XIAP?COMMD1?ATP7B and ATP7 A in both cells are not seriously affected.3.Effect on ATP7 B after inhibiting the expression of COMMD1Use si RNA to interfere with the expression of COMMD1.Compared to scramble si RNA control group,the extent of ATP7 B protein's decrease of the interference group after exposing to lead goes down significantly(p<0.05),while the level of ATP7 A protein doesn't change much(p>0.05).4.Effect on copper level in RBE4 and Z310 cells after inhibiting the expression of COMMD1Use si RNA to interfere with COMMD1's expression.Compared to scramble si RNA control group,the extent of copper level's increase of the interference group has a significant reduction after exposing to lead.(p<0.05)5.Highly expressed XIAP's effect on COMMD1 and ATP7BConstruct plasmids that highly express XIAP.Compared to Vector group,highly expressed XIAP group which exposes to lead is significantly inhibited in induction of COMMD1 protein and down-regulation of ATP7 B level(p<0.05),while it is not apparently affected with ATP7 A level(p>0.05).6.Highly expressed XIAP's effect on copper level of REB4 and Z310 cellsConstruct the plasmid to highly express XIAP.Compared to Vector group,highly expressed XIAP group which exposes to lead is significantly dropped in the increase of intracellular copper level.(p<0.05)7.Study of functional domain of XIAP which regulates expression of COMMD1Transfect cells with wild type plasmid and the mutant type plasmid with E3 ubiquitin ligase site separately.Compared to the control group,the experimental group which highly expresses wild type XIAP has a significant decrease in protein level of COMMD1(p<0.05),while the other experimental group which highly expresses mutant XIAP type with E3 ubiquitin ligase site has no big difference in protein level of COMMD1(p>0.05).Conclusions:1.Lead exposure can cause increase in copper level of BBB and its cells by down regulating the protein level of copper transporter ATP7 A and ATP7 B.2.Lead exposure can decrease the expression of XIAP protein,increasing COMMD1's protein level at its downstream,thus down regulating protein level of ATP7 B specifically,resulting in blockage of intracellular copper efflux and intracellular copper accumulation.3.The regulation of XIAP-COMMD1-ATP7 B is at protein level.COMMD1 is degraded by RING domain of XIAP with E3 ubiquitin ligase activity.