Role Of TMEM187 In Mitochondrial Iron Metabolism

Background:Iron is an essential element for all living organisms.An excess of iron may participate in the formation of reactive oxygen species(ROS)via Fenton reaction to further result in oxidative damage in cells.Thus,iron uptake,export,storage,and utilization are highly regulated.Disturbed iron homeostasis is associated with some diseases.Iron in the circulatory system binds to transferrin(Tf).Cellular iron uptake is mainly mediated via endocytosis of iron-laden Tf-transferrin receptor 1(TfR1)complex on the cell surface and regulated by iron regulatory protein 1 and 2(IRP1 and IRP2),two cytosolic iron-sensing proteins.IRP1 and IRP2 post-transcriptionally regulate the expression of iron-related proteins by binding to the iron responsive element(IRE)in response to iron.Under low iron circumstances,IRP1/2 bind to IRE in the 5'-untranslated region(UTR)of ferritin to inhibit its translation,whereas they bind to the IREs in the 3'-UTR of TfR mRNA to stabilize the mRNA and facilitate its translation.Thus,iron uptake is favored.Under high iron circumstances,IRP1 gains function as a cytosolic aconitase and its IRE-binding activity is lost,while IRP2 degrades through iron-dependent pathway by the hemerythrin-like ubiquitin ligase,FBXL5.Therefore,iron repletion decreases the rate of cellular iron uptake and increases the rate of iron sequestration.After entering a cell,iron can be directly utilized,blended into labile iron pool(LIP),sequestered in ferritin,or transported into mitochondrion.Mitochondria are central for energy production(ATP)and regulation of cellular iron metabolism.The majority of iron imported into cells are utilized within this organelle for synthesis of heme and iron sulfur clusters.Very little is known about intracellular iron transport pathways that facilitate mitochondrial iron import.Iron metabolic disorder is followed by ROS production,which will impair mitochondrial function and promote cell apoptosis.Thus,it is important to make certain refinement of mitochondrial iron metabolism and regulation.TMEM187 is membrane protein with 6 transmembrane domains and appears to be ubiquitously expressed.It does not seem to be remarkably conserved in evolution.Though cytoplasm,nucleoli,golgi,endoplasmic reticulum(ER),or ER-golgi localizations was predicted or evidenced,the exact subcellular localization of TMEM187 has not yet been confirmed.Previous study has demonstrated that overexpression of TMEM187 caused rounded cell,dented nuclei and apoptosis.However,the exact function has not been described.The purpose of this study is to clarify TMEM187 subcellular localization and characterize the function of the protein in mitochondrial iron metabolism.Objectives:The aims of this study are:1)to determine TMEM187 subcellular localization;2)to investigate whether TMEM187 was involved in mitochondrial iron metabolism and the mechanism how it occurs;3)to reveal what phenotype the abnormal expression of TMEM 187 will result in and the mechanism involved.Methods:Human TMEM187 was amplified by polymerase chain reaction(PCR)and ligated with pcDNA3.1(-)-myc and pEGFP-N1 vectors,respectively,to construct a recombinant plasmids pcDNA3.1(-)-TMEM187-6×myc and pEGFP-TMEM187.The positive and negative control plasmids,pcDNA3.1(-)-TMEM 187-6×myc,and pEGFP-TMEM187 were transiently transfected into HEK293 cells.TMEM187 subcellular localization was determined under a confocal microscope.Cells expressing TMEM 187-myc were harvested,Western blotting was used to estimate the expression levels of iron related proteins.Ferrozine assay,rhodamine B-[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester(RPA),calcein-AM,and Perl's staining were performed to evaluate cellular,mitochondrial,and cytosolic iron contents.Fluorescence resonance energy transfer(FRET)and Co-immunoprecipitation(Co-IP)were used to estimate the interaction of TMEM 187 and MFRN.siRNAs were used to knock down TMEM187,MFRN1 or MFRN2.Cell lysates were obtained to determine ATP levels,apoptosis index,oxidative stress,and the activities of xanthine oxidase(XOD),aconitase,superoxide dismutase(SOD),and electron-transport chain complexes,respectively.IRE binding activities of IRPs were measured by electrophoretic mobility shift assay(EMSA).Results:1)Human TMEM187 is mainly localized in golgi as an integral membrane protein.2)Expression of TMEM187 triggered mitochondrial and cytosolic iron accumulation,though,and activated the iron starvation signaling,stimulated the expression of TfRl and ferritin,so that iron uptake and storage increased and cellular iron homeostasis was disturbed.3)Unexpectedly,knockdown of human TMEM187 also promoted mitochondrial iron uptake.4)The effect of human TMEM187 on mitochondrial iron metabolism was mediated through MFRN1/2,i.e.,knockdown of TMEM187 increased the mitochondrial iron by upregulation of MFRN1 and overexpression of TMEM187 increased mitochondrial iron through upregulation of MFRN2.Very strikingly,TMEM187 possibly physically interacts with MFRN2.5)Human TMEM187 was functional in mouse cells,which have no TMEM187 ortholog,in a same way as in human cells.6)Overexpression of human TMEM187 decreased cellular ability of scavenging ROS and disrupted cellular redox state,resulting in cellular oxidative stress and mitochondrial damage.7)Overexpression of human TMEM187 promoted the cell apoptosis and inhibited tumor formation in nake mice.Conclusion:Golgi membrane protein TMEM187 plays an important role in cellular and mitochondrial iron metabolism through regulating expression of mitochondrial inner membrane proteins MFRN1 and MFRN2;TMEM187 is a potential pro-apoptotic factor.