| Parkinson’s disease(PD)is a common neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra(SN),the formation of intracytoplasmic inclusions known as Lewy bodies(mainly including abnormal alpha-synuclein aggregation)in residual dopaminergic neurons and depletion of dopamine levels in the striatum.Clinically,the disease is attended by a constellation of disabling motoric deficits including resting tremor,rigidity and postural instability,accompanied by nonmotor symptoms,such as olfactory decline,neuropsychiatric symptoms,gastrointestinal disorders,and cognitive impairment.Genetic,non-genetic factors and aging all contribute to the pathogenesis of PD.Oxidative stress,mitochondria dysfunction,iron deposits,inflammation,aberrant protein aggregation,apoptotiosis,excessive iron deposition etc.,are involved in the demise of nigral dopaminergic neurons.Neuropathological,neurochemical and neuroimaging studies demonstrated iron was selectively deposited in the SN and elevated iron was observed in the surviving dopaminergic neurons in the postmortem PD human brains.This indicates iron metabolic disturbance is of vital importance of dopaminergic neurons degeneration in PD.Intracellular iron levels are balanced by two orthologous iron regulatory proteins(IRPs),namely IRP1 and IRP2,that coordinate the proteins posttranscriptionally involved in the iron uptake,utilization,storage,and export by binding to cis-regulatory iron responsive elements(IREs)in the untranslated region of respective m RNAs.It had been found that IRP2 knockout mice displayed neurodegenerative symptoms and nigral iron deposition,indicating IRP2 is a key factor in regulating the brain iron homeostasis.Our previous study also showed decreased IRP2 levels in 1-methyl-4-phenylpyridinium ion(MPP+)-treated MES23.5 cells,as well as in the SN of homozygous A53 T PD mice at 6-month age.This indicated that IRP2 might participate in the pathological process of iron metabolism disorder in PD,although the underlying mechanisms were largely unknown.IPR2 activity and stability must be exquisitely regulated in the context of brain iron homeostasis.Besides intracellular iron concentration,hypoxia,oxidative stress,inflammatory response,vitamin A,ascorbic acid and nitric oxide can affect the expression of IRP2.In addition to the transcriptional regulation and protein synthesis,the post-translational modification is a major pathway to maintain the stability of the protein and eventually the protein levels.As a kind of important form of protein modifications,ubiquitin and deubiquitin jointly maintain the stability of the protein.E3 ubiquitin-ligase enzymes and deubiquitinase(DUB)are two key enzymes involved in the reversible processes of ubiquitination and deubiquitination.Even though two E3 ubiquitin ligases that promote IRP2 polyubiquitylation for degradation have been found,the DUBs of IRP2 have not been identified.In view of ubiquitin and deubiquitin in maintaining the IRP2 protein levels,implying these mechanisms helps to reveal how IRP2 protein levels are maintained,as well as how this contributes to the demise of dopaminergic neurons in PD.In the present study,a whole DUB proteome-wide screening was performed to find the indicated DUB of IRP2.We found that OTU domain-containing protein 3(OTUD3)specifically up-regulated IRP2 levels by maintaining IRP2 stability.In order to further elucidate the molecular mechanisms of OTUD3 on IRP2 regulation as well as its role in the nigral iron deposition in PD,we further investigated whether and how OTUD3 interacted with IRP2.Then,we elucidated the effects of OTUD3 on ubiquitination level of IRP2,and determined the ubiquitination linkage of IRP2 regulated by OTUD3.We also used OTUD3 knockout mice model to study the role of OTUD3 and IRP2 ineraction in nigral iron deposition.In details,we evaluated the changes of colonic motility by one-hour stool characteristics and frequency assay.And rotarod test,pole test and Cat Walk analysis were applied to estimate motor coordination ability.Using immunofluorescent staining,we observed the number of the nigral dopaminergic neurons of SN.High-performance liquid chromatography with electrochemical detection was employed to detect dopamine and its metabolites in the striatum.Inductively coupled plasma mass spectrometer(ICP-MS)was used for the determination of the iron content of SN.Results as follows:1.OTUD3 maintained IRP2 stability(1)To identify whether DUB was responsible for IRP2 stability,we performed a whole DUB proteome-wide screening.Each DUB in the human proteome consisting of six families-USPs(ubiquitin-specific proteases),UCHs(ubiquitin C-terminal hydrolases),OTUs(ovarian tumour proteases),MJDs(Machado-Joseph disease related enzymes),JAMMs(Josephins and JAB1/MPN/Mov34 metalloenzymes)and MCPIP(monocyte chemotactic protein-induced protein)was overexpressed in HEK293 T cells and the expression of endogenous IRP2 was analysed.This screening eventually identified that OTUD3 remarkably upregulated IRP2 levels.(2)Increasing amounts of OTUD3 plasmids but not OTUD3C76 A plasmids in the HEK293 T cells resulted in IRP2 upregulation in a dose-dependent manner.Depletion of OTUD3 by a short interfering RNA(siRNA)pool markedly decreased IRP2 levels,and two independent OTUD3 siRNAs both showed significant effects.Protein levels of endogenous IRP2 increased gradiently in HEK293 T cells with the increased transfection of gradient Myc-OTUD3.However,protein levels of endogenous IRP2 did not increase when HEK293 T cells were transfected with increasing amounts of OTUD3 enzyme activity center mutant Myc-OTUD3C76 A.The expression levels of endogenous OTUD3 and IRP2 were significantly decreased in OTUD3 siRNA group compared with the control siRNA group in HEK293 T cells.(3)In HEK293 T cells with OTUD3 knockdown by OTUD3 siRNA,transfection by MycOTUD3,rather than Myc empty vector or Myc-OTUD3C76 A restored the protein level of IRP2.However,when 20 μmol/L proteasome inhibitor MG132 was added for 8 hrs in HEK293 T cells transfected with OTUD3 siRNA,the protein levels of IRP2 were restored compared with the OTUD3 siRNA transfected group without MG132 treatment.(4)Compared with the empty vector group in HEK293 T cells,real time PCR showed no significant changes in m RNA level of IRP2 in the Myc-OTUD3 transfected group.No obvious changes were found in m RNA levels of IRP2 between OTUD3 siRNA group and OTUD3 siRNA control group in HEK293 T cells.(5)To prove that OTUD3 affects IRP2 stability per se,cells were treated with the protein synthesis inhibitor cycloheximide.Compared with the empty vector group in HEK293 T cells,the half-life of IRP2 protein in Myc-OTUD3 group was obviously prolonged.Whereas,the half-life of IRP2 protein in OTUD3 siRNA group was obviously shortened than the control siRNA group in HEK293 T cells.2.OTUD3 interacted with IRP2(1)Flag empty vector/Myc-OTUD3 and Flag-IRP2/Myc-OTUD3 were transfected in HEK293 T cells,respectively.The IRP2 was immunoprecipitated in Flag-IRP2/MycOTUD3 group,whereas,not in Flag empty vector/Myc-OTUD3 group.On the contrary,Myc empty vector/Flag-IRP2 and Myc-OTUD3/Flag-IRP2 were transfected in HEK293 T cells,respectively.The OTUD3 was immunoprecipitated in MycOTUD3/Flag-IRP2 group,but not in Myc empty vector/Flag-IRP2 group.The results indicated OTUD3 interacted with IRP2.(2)HEK293T cells were labeled with endogenous antibodies of IRP2 and OTUD3,and it was found that both IRP2 and OTUD3 were mainly colocalized in the cytoplasm of HEK293 T cells.The results were also verified in SH-SY5 Y cells.3.OTUD3 de-polyubiquitylated IRP2 and effectively removed K48-linked polyubiquitylation(1)Myc empty vector/HA empty vector,Myc empty vector/HA-ub,Myc-OTUD3/HA-ub were transfected into HEK293 T cells,respectively.Compared with Myc empty vector/HA empty vector group,ubiquitination of IRP2 was detected in Myc empty vector/HA-ub group.Compared with Myc empty vector/HA-ub group,the ubiquitination level of Myc-OTUD3/HA-ub group was significantly decreased.This indicated that overexpression of Myc-OTUD3 could remove IRP2 ubiquitination.With the administration of 100 μg/m L ferric ammonium citrate(FAC)supplement in the above three groups,ubiquitination of IRP2 was enhanced in FAC group and OTUD3 could remove the enhanced IRP2 ubiquitination caused by FAC.In addition,control siRNA and OTUD3 siRNA was transfected into HEK293 T cells,respectively.Compared with the control siRNA group,ubiquitination of IRP2 was increased in two OTUD3 siRNA sequences(#1 GGCCAGCCCUAGUGAAGAA,#2 GAAAUCAGGGCUUAAAUGA)group.The results showed that knockdown of OTUD3 increased ubiquitination of IRP2.(2)Myc empty vector/HA empty vector/His empty vector,Myc empty vector/HA empty vector/His-ub,Myc empty vector/HA-HOIL1/His-ub and Myc-OTUD3/HAHOIL1/His-ub were transfected in HEK293 T cells,respectively.Compared with Myc empty vector/HA empty vector /His empty vector group,ubiquitination was detected in Myc empty vector/HA empty vector/His-ub group.Compared with Myc empty vector/HA empty vector/His-ub group,the ubiquitination level of IRP2 increased slightly in Myc empty vector/HA-HOIL1/His-ub group.Compared with Myc empty vector/HA-HOIL1/His-ub group,the ubiquitination level of IRP2 was significantly decreased in Myc-OTUD3/HA-HOIL1/His-ub group.This suggested that overexpression of Myc-OTUD3 could remove the ubiquitination of IRP2 ubiquitinligase HOIL1.When the above HA-HOIL1 was replaced by HA-FBXL5,it revealed that OTUD3 could also remove the ubiquitination caused by FBXL5.(3)We next investigated which type of ubiquitin chain of IRP2 was affected by OTUD3.Myc empty vector/HA empty vector,Myc empty vector /WT HA-ub,Myc-OTUD3/WT HA-ub,Myc empty vector/K0 HA-ub,Myc-OTUD3/K0 HA-ub,Myc empty vector/K48 HA-ub,Myc-OTUD3/K48 HA-ub,Myc empty vector/K63 HA-ub and Myc-OTUD3/K63 HA-ub were transfected into HEK293 T cells,respectively.Compared with Myc empty vector/WT HA-ub group,the ubiquitination level of IRP2 in Myc-OTUD3/WT HA-ub group was decreased.Compared with Myc empty vector/K48 HA-ub group,the ubiquitination level of Myc-OTUD3/K48 HA-ub group was decreased.However,there was no change between Myc empty vector/K0 HA-ub and Myc-OTUD3/K0 HA-ub,Myc empty vector/K63 HA-ub and Myc-OTUD3/K63 HA-ub.The results suggested that OTUD3 effectively removed Lys 48-linked polyubiquitylation,but not monoubiquitylation or the non-degradative Lys 63-linked polyubiquitylation of IRP2.4.OTUD3-/-mice of 24-month old exhibited motor and non-motor PD symptoms(1)In the rotarod test,the residence time of mice on rotarod treadmills was 36.1% shorter in OTUD3-/-mice of 24-month old compared with wide type(WT)mice.OTUD3-/-mice of 24-month old showed a significant 83.0% increase in the time to turn to orient down-ward and 42.0% increase in total time in the pole test,compared with WT mice.In Catwalk analysis,max contact area and print area of right hindpaw,left hindpaw and left forelimb,print length and stride length of four limbs,print width of forelimb decreased in OTUD3-/-mice of 24-month old.Step cycle of right forelimb,left forelimb and left hindpaw delayed.The swing speed was decreased,as indicated by a concomitant increase of the stand.Meanwhile,regularity index was decreased and the step sequence was changed.(2)Using immunohistochemistry assay,we observed the number of TH-positive neurons was decreased by 22.6% in the SN in OTUD3-/-mice of 24-month old.The contents of HVA in the striatum of OTUD3-/-mice of 24-month old were decreased by 59.3% compared with WT mice.No differences were detected in dopamine contents,although there was a downward tendency.(3)The colonic motility was observed in OTUD3 and WT mice of 24-month old.In OTUD3-/-mice,wet weight of stool,dry weight of stool and stool frequency were lower than WT mice by 48.7%,44.5% and 43.0%,respectively,although no changes were observed in water content of stool.5.Deletion of OTUD3 caused iron metabolism disorders in both mice and cells(1)Iron content was elevated approximately 2 folds in the SN in OTUD3-/-mice of 24-month old.The levels of IRP2 and transferrin receptor 1(Tf R1)were decreased,however,the levels of ferritin were elevated in the SN of OTUD3-/-mice of 24-month old compared with WT mice.(2)Compared with WT mice,the levels of Tf R1 were decreased and the levels of ferritin were increased in mouse embryonic fibroblast(MEF)cells in OTUD3-/-mice.Although ferrous iron influx was unchanged,iron content was increased by 33.9% in MEF cells of OTUD3-/-mice.Intracellular iron deposit was found by Prussian blue staining in MEF cells of OTUD3-/-mice.By flow cytometry,we observed a decrease of ΔΨm(24.2%)and an elevation of ROS(39.2%)in MEF cells of OTUD3-/-mice.The expression of SOD1 and Bcl-2/Bax was decreased in MEF cells of OTUD3-/-mice.Furthermore,apoptosis rate was increased by 90.3% in MEF cells of OTUD3-/-mice.(3)Myc empty vector and Myc-OTUD3 were transfected in SH-SY5 Y cells for 48 hrs,respectively.Compared with Myc empty vector group,protein levels of IRP2 and Tf R1 were increased,and ferritin was decreased in Myc-OTUD3 group.Control siRNA and OTUD3 siRNA were transfected in SH-SY5 Y cells for 48 hrs,respectively.Compared with control siRNA group,protein levels of IRP2 and Tf R1 were decreased,and ferritin was increased in OTUD3 siRNA group.This study reported OTUD3 remarkably up-regulated IRP2 protein levels in a dosedependent manner,without affecting transcription levels.OTUD3 maintained IRP2 stability through the ubiquitin-proteasome pathway,depending on the deubiquitylase activity.In addition,OTUD3 could interact with IRP2.We further demonstrated OTUD3 could remove IRP2 ubiquitylation.Following colonic motility and motor features,OTUD3-/-mice performanced a loss of the dopaminergic neurons in the SN and HVA in the striatum accompanied with iron deposits in the SN.In addition,iron contents increased in the SN of OTUD3-/-mice.Besides,OTUD3-/-MEFs showed iron metabolism related protein dysregulations,elevated iron contents and iron deposits,embraced by aggravagated ROS production,diminished SOD1 and induced mitochondrial apoptosis.Iron metabolism related protein dys-regulations were also found in SH-SY5 Y cells with overexpression or deletion OTUD3.Taken together,this study identified OTUD3 was the first DUB of IRP2,and uncovered the mechanisms of IRP2 regulated by OTUD3.OTUD3 is a molecule that has not been fully studied,which is limited to its role in stabilizing phosphatase and tension homologue deleted on chromosome 10(PTEN)protein that participates in the development of tumor.In this study,OTUD3 was involved in the regulation of IRP2,so as to participate in the abnormal regulation of iron metabolism in PD,providing novel perspectives for the better understanding of OTUD3.In present study,OTUD3-/-mice exhibited motor and non-motor PD symptoms,which provided a new phenotype for OTUD3-/-mice. |
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