| IntroductionRetinoids represent a popular group of differentiation inducers which are successfully used in cancer treatment.The differentiation inducing activity of retinoids is primarily mediated by retinoic acid receptors(RARs),which work as selective ligand-activated transcription factors.At the same time,retinoids could lead to ubiquitin-proteasome pathway mediated degradation of RARs,which limits the efficacy and clinical use of retinoic acid.RARs consist of α,β and γ three types.Regulation of gene transcription by retinoids is typically caused by constitutive binding of RAR-RXR heterodimers to specific retinoic acid response elements(RAREs)found in the promoter regions of target genes.With further study,posttranslational modification were found to play an important role in the activation and degradation of RARs induced by RA agonist.Protein S-palmitoylation is a reversible posttranslational modification with fatty acids mediated by protein acyltransferases(PATs)DHHC family,which has an importent role in regulating protein location,trafficking and function.Recent study found its important role in regulating essential cellular functions.Protein palmitoylation modification usually occurs on cysteine residues of conservative transmembrane domains.Many databases could predict palmitoylation modification sites of proteins based on reported protein sequences.The prediction results of Css-Palm and NBA-Palm suggest that RARα could be palmitoylated.Therefore,whether and how RARa undergoes S-palmitoylation and the role of S-palmitoylation in RARα transcriptional activity are intriguing avenues of investigation which may provide new molecular mechanisms and targets for cancer differentiation therapy.Section 1 S-palmitoylation of RARa and its regulatory mechanismObjectiveRetinoids can regulate the proliferation and differentiation of cells by activating RARa.And posttranslational modification were found to play an important role in the activation and degradation of RARs induced by RA agonist.Protein S-palmitoylation is a reversible posttranslational modification with fatty acids,which have an important role in regulating cellular functions.The aim of this study was to investigate whether and how RARα undergoes S-palmitoylation.MethodsWe used a metabolic labeling strategy using an alkyne-tagged palmitic acid analogue(ALK 14)to test the S-palmitoylation occurs on RARα.Cells were cultured in the presence of ALK 14,proteins were immunoprecipitated and conjugated to Cy5.5-azide or Biotin-azide via click chemistry,and the fluorescence signal was imaged afterSDS-PAGE;The potential palmitolation sites of RARα were determined by click chemistry assay when cystein were replaced with serine;Hydroxylamine were used to determine cysteine S-palmitoylation;We used co-immunoprecipitation to identify the potential protein acyltransferases(PATs)responsible for RARa palmitoylation;We manipulated the potential PATs levels by shRNA,and then determined the palmitoylation level of RARα.ResultsPalmitoylation of RARα:① Overexpressed RARα could be palmitoylated.Cells were cultured in the presence of ALK 14,proteins were immunoprecipitated and conjugated to Cy5.5-azide or Biotin-azide via click chemistry,and the fluorescence signal was imaged after SDS-PAGE.And RARa had fluorescent labeling,suggesting that it contains fatty acylation;② S-palmitoylation was found on RARα:After the samples were treated with 0.8 M hydroxylamine,most of the fluorescence signal on RARa was removed,confirming that RARα has cysteine S-palmitoylation.③ S-palmitoylation was found on endogenous RARα:U2OS cells were labeled by ALK 14,and click chemistry assay results showed that RARa could be palmitoylated.④Cys-8,Cys-91 and Cys-143 of RARa could be palmitoylated.To further confirm palmitoylation occurs on cysteine residues,we mutated Cys-8,Cys-91 and Cys-143 to serine.We observed a significant decrease in the fluorescence signal of the cysteine mutants C8S,C91S,C143S and C91/143S.⑤ RARa is a DHHC3 target:We co-overexpressed HA-tagged DHHCs with flag-tagged RARα,and the results of immunoprecipitation experiment showed that RARα had an interaction with DHHC3 and DHHC21;We further knockdowned DHHCs which had interaction with RARα,examined the fatty acylation level using the ALK 14 metabolic labeling approach.Only knockdown of DHHC3,reduced RARa palmitoylation level.At the same time,co-expressed DHHC3 could enhance RARa palmitoylation level,while co-expressed catalytically inactive mutant DHHC3-C157S reduced RARa palmitoylation level.These results suggest that DHHC3 is required for the S-palmitoylation RARα.ConclusionsWe show that RARa undergoes S-palmitoylation on three cysteine residues,Cys-8,Cys-91 and Cys-143.We further identified DHHC3 as a RARα palmitoylating enzyme.What’s more,DHHC21 have an interaction with RARα.Section 2 Study on the role and mechanism of RARa S-Palmitoylation via DHHC3 in osteosarcoma differentiationObjectiveRetinoic acid receptor α(RARα)are classically considered as nuclear ligand-dependent regulators of transcription,which regulates the expression of target genes involved in cell growth and differentiation.As we found that RARa could be palmitoylated by DHHC3 in section 1.Here,we will find out the physiological function of RARα S-palmitoylation,and we will further investigate the role of RARa S-palmitoylation in osteosarcoma differentiation therapy induced by ATRA.MethodsRARE-luciferase assay and Q-RT-PCR assay were used to detect the RARαtranscriptional activation,when we mutated Cys-8,Cys-91 and Cys-143 to serine;Protein expression level of RARα was determined by Western Blotting,when treated with CHX,RARa agonist or RARα antagonist;Co-mmunoprecipitation was performed to detect the interaction between RARa and RXRα;RARE-luciferase assay and Q-RT-PCR assay were used to detect the RARα transcriptional activation,when DHHC3 is down regulated or treated with 2BP;Osteosarcoma differentiation induced by ATRA were determined by alkaline phosphatase staining and Q-RT-PCR.Results1)The role of RARa S-palmitoylation in its transcription:① The effect of palmitoylation sites mutation on RARa transcription induced by ATRA:Three palmitoylation sites(Cys-8,Cys-91 and Cys-143)of RARα were replaced by serine,and RARE luciferase assay and Q-RT-PCR(mRNA level of RARB,CYP26A1,EGR1,TRAIL and UBE1L)results revealed that the C91S,C143S and C91/143S mutants clearly impaired RARα transcription activity induced by ATRA.② Depletion of DHHC3 impaired RARα transcription activity induced by ATRA.Q-RT-PCR results revealed that depletion of DHHC3 in U2OS cells impaired RARa ranscription activity induced by ATRA.③ Palmitoylation inhibitor 2BP impaired RARa transcription activity induced by ATRA.RARE luciferase result showed that RARE activity induced by ATRA were downregulated from 2.49 ± 0.19 to 1.22±0.10(p<0.001)by treating with 2BP.2)RARa S-palmitoylation affects protein stability induced by RARa agonist:RARa agonist could lead RARa to ubiquitin-proteasome pathway mediated degradation when activated RARα.① The protein level increased when C91,C143 and C91/143 mutated.Three palmitoylation sites(Cys-8,Cys-91 and Cys-143)of RARα were replaced by serine,Western Blotting results showed that C91S,C143S and C91/143S could increase the protein level of RARα;The difference of protein expression were abolished when treated with proteasome inhibitor MG 132;C91S,C143S and C91/143S degraded slowly when treated with CHX,ATRA and AM80;C91S,C143S and C91/143S accumulated slowly when treated with Ro41-5253;② Depletion of DHHC3 increased RARa stability.The protein level of RARa was increased upon DHHC3 depletion;Depletion of DHHC3 enhanced the stability RARa when treated with ATRA or CHX.③ Palmitoylation inhibitor 2BP increased RARα stability:2BP promoted the accumulation of RARα;2BP enhanced the stability RARa when treated with ATRA or CHX.3)The mechanism of RARα S-palmitoylation regulating transcription activity:① The RARα/RXRα interaction was confirmed by coimmunoprecipitation(CO-IP);The effect of palmitoylation sites mutation on the formation of RARα/RXRα heterodimer:C91S,C143S and C91/143S impaired the formation of RARα/RXRα heterodimer.② The effect of 2BP on the formation of RARα/RXRα heterodimer:The formation of RARα/RXRα heterodimer was impaired by 2BP.4)RARα palmitoylation regulated osteosarcoma differentiation induced by ATRA.① The data of ALP and Q-RT-PCR assay showed that C91S,C143S and C91/143S impaired the osteosarcoma differentiation induced by ATRA.② The data of ALP assay showed that 2BP could impaied the osteosarcoma differentiation induced by ATRA.ConclusionThese results,thus,briefly suggested palmitoylation may potentially play an synergetic role to transactivation of RARα via regulating RARα/RXRα heterodimer,RARa palmitoylation is essential for osteosarcoma differentiation induced by ATRA.Section 3 DHHC21 regulates AML cell differentiation via RARαObjectiveDHHC21 was found to interact with RARα,while didn’t affect the palmitoylation level of RARa in section 1.We will further investigate the effect of DHHC21 on the function of RARα.Further more,we will explore the differentiation-blocking role of DHHC21 in AML cells.MethodsRARE-luciferase assay was used to detect the RARα transcriptional activity upon the deleption of DHHC21;The total cell number upon the deleption of DHHC21 were determined by manual counting in Burker chambers;The AML cell differentiation was determined by assessing cell surface marker CD 11b expression,NBT reduction assay as well as morphological changes;Western Blotting and Q-RT-PCR were used to determine the protein and mRNA of RARβupon the deleption of DHHC21.Results1)The effect of DHHC21 deleption on transcriptional activity.The data of dual-luciferase assay showed that DHHC21 could upregulate the transcriptional activity of RARα.2)Knockdown of DHHC21 displayed the anti-cancer activity by inducing AML cells differentiation.All three shRNA(shDHHC21#1,#2 and#3)could inhibit cell proliferation.The depletion of DHHC21 induced myeloid differentiation in human AML cells,by increasing CD11b expression,NBT reduction rate as well as mature morphology of neutrophil.3)The mechanism of DHHC21 knockdown in AML cells differerntiation:Knockdown of DHHC21 has a profound activation effect on RARa pathway.4)Palmitoylation inhibitors displayed the anti-cancer activity by inducing AML cells differentiation.2BP induced myeloid differentiation in human AML cells,by increasing NBT reduction rate and with an increased cytoplasm-to-nuclear ratio.Flow cytometry was used to detect the expression of surface marker CD11b and CD14,and the results showed that 2BP could induce the differentiation of CD 14 positive monocytes.These results suggest that palmitoylation inhibitors can take part in the differentiation of AML.ConclusionWe provide evidence that the depletion of DHHC21 overcomes the myeloid differentiation blockade of AML cells by activating RARα.We further demonstrate that the administration of palmitoylation inhibitors enables the myeloid differentiation of AML cells.Together,our findings offer new insights into the role of DHHC21 in inducing differentiation therapy of AML cells and suggest that DHHC21 as well as palmitoylation may be a novel differentiation therapeutic target for AML leukemia. |
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