DEC1 Represses Cardiomyocyte Hypertrophy By Recruiting PRP19 As An E3 Ligase To Promote Ubiquitination-Proteosome-Mediated Degradation Of GATA4

Part Ⅰ Screening and Identification of Endogenous Inhibitors for Pro-hypertrophic Transcription Factor GATA4Objective: To screen candidate molecules for GATA4(GATA binding protein 4)protein endogenous inhibitors by interacting protein mass spectrometry technique and preliminarily investigate the effect of candidate molecules on GATA4 protein activity.Methods: Establishing in vitro pathological cardiomyocyte hypertrophy model by stimulating rat cardiac myoblast cell lines H9C2 cells using 50 μM phenylephrine(PE),equivalent dose of phosphate buffer solution(PBS)as control,exploring candidate molecules for GATA4 endogenous inhibitors among GATA4 interacting proteins by mass spectrometry technique.Constructing expression plasmids of candidate molecules for GATA4 endogenous inhibitors and screening out the potent endogenous inhibitor differentiated embryo chondrocyte 1(DEC1)for GATA4 protein activity by dual-luciferase reporter gene assay.Constructing expression plasmids of DEC1 and GATA4 with different domain truncation and verifying the interaction of DEC1 and GATA4 protein and specific domain for the interaction.Results: Three candidate molecules for GATA4 endogenous inhibitors were screened out by analyzing interacting protein mass spectrometry data,which included HNRNPU,DEC1 and RSL1D1L1 interacting with GATA4 protein only in PBS group in two independent experiments.DEC1,but not HNRNPU and RSL1D1L1,significantly inhibited GATA4 protein activity in dose-dependent manner.DEC1 and GATA4 could mutually combine,b HLH domain and ZNII domain were responsible for the interaction.Conclusions: DEC1 could restrain GATA4 protein activity,b HLH domain of DEC1 and ZNII domain of GATA4 mediated the combination of the both.Part Ⅱ The Role of DEC1 in Pathological Cardiomyocyte Hypertrophy and Cardiac HypertrophyObjective: To establish in vitro pathological cardiomyocyte hypertrophy cell model and in vivo pathological cardiac hypertrophy animal model and to explore the role of GATA4 endogenous inhibitor DEC1 in pathological cardiomyocyte hypertrophy and cardiac hypertrophy.Methods: Constructing adenoviruses of overexpressing(AdDec1)and knock down(Adsh Dec1)Dec1 and infecting neonatal rat ventricular myocytes(NRVM)and H9C2 cells,AdGfp and Adsh Scr were used as control adenoviruses,which had no effect on DEC1 expression.PE was used to stimulate H9C2 and NRVM cells to establish an in vitro cell model of pathological cardiomyocyte hypertrophy.Western Blotting,Real-Time PCR and cell immunofluorescence assays were used to evaluate the effects of enhanced and reduced Dec1 function on cardiomyocyte hypertrophy.Transverse aortic constriction(TAC)was performed in systemic Dec1 knockout(Dec1-KO)mice to induce pathological cardiac hypertrophy,wild type(WT)littermates were used the control mice.The sham group(Sham)was wrapped silk threads around the aortic arch without ligation.The mice heart tissues were obtained after TAC operation for 4 weeks,the heart weight to body weight(heart weight/body weight,HW/BW)ratio and the heart weight to tibial length(heart weight/tibial length,HW/TL)ratio were measured.Hematoxylin-eosin(HE)staining and wheat germ agglutinin(WGA)staining were used to observe myocardial hypertrophy and cardiomyocyte hypertrophy in mice.Western Blotting was used to detect the protein expression of cardiac hypertrophy marker molecules ANP,MYH7 and GATA4 in the heart tissues in each group.Using adenoviruses Adsh Gata4 and/or Adsh Dec1 to infect NRVM cells,detecting the effects of Gata4 knockdown and/or Dec1 knockdown on PE-stimulated NRVM cells.Results: The in vitro experiments showed that ANP and GATA4 protein levels were significantly reduced after Dec1 overexpression,but increased after Dec1 knockdown.Dec1 overexpression in NRVM and H9C2 cells could significantly down-regulate the expression of cardiac hypertrophy markers Nppa and Nppb m RNA,while knockdown of Dec1 significantly up-regulated the expression of Nppa and Nppb m RNA.The cell immunofluorescence assay indicated that Dec1 overexpression could inhibit NRVM cells hypertrophy stimulated by PE,while knockdown of Dec1 could promote that of NRVM.Dec1 gene knockout could significantly aggravate the pathological myocardial hypertrophy induced by TAC,and promote the further up-regulation of ANP,MYH7 and GATA4 proteins after TAC.Knockdown of Gata4 could significantly inhibit the effect of Dec1 silencing on PE-induced NRVM cells hypertrophy,and inhibit the further up-regulation of Nppa and Nppb m RNA expression in NRVM cells after Dec1 knockdown.Conclusions: DEC1 could significantly inhibit pathological cardiomyocyte hypertrophy and cardiac hypertrophy.The inhibitory effect of DEC1 on pathological cardiomyocyte hypertrophy was dependent on its inhibiting effect on GATA4 protein activity.Part Ⅲ Exploration of Molecular Mechanism for DEC1 Suppressing GATA4 Protein ActivityObjective: To explore the molecular mechanism of DEC1 inhibiting GATA4 protein activity by examining GATA4 protein level,phosphorylation level of GATA4 serine 105(p-GATA4Ser105),nuclear and cytoplasmic distribution of GATA4 and GATA4 ubiquitination level as well as ubiquitination type using in vivo and in vitro ubiquitination assay.Methods: Infecting NRVM and H9C2 with AdGfp and AdDec1 and establishing PE-induced cardiomyocyte hypertrophy,detecting the effect of DEC1 on Gata4 m RNA level by Real-Time PCR assay,examining p-GATA4Ser105 level and total GATA4 level in H9C2 and NRVM cells by Western Blotting,investigating the nuclear and cytoplasmic distribution of GATA4 in H9C2 cells by nucleus-cytoplasm separation assay.Treating H9C2 and NRVM cells with 50 μM proteasome inhibitor MG132,10 μM lysosomal inhibitor chloroquine(CQ),and equivalent dose of PBS,exploring the effect of MG132 and CQ on DEC1-mediated GATA4 protein degradation.Transfecting 293 T cells with GATA4,DEC1,and different types of ubiquitin expression plasmids,examining GATA4 ubiquitination level and ubiquitination type by DEC1 through co-IP and Western Blotting assay.Transfecting 293 T cells with GATA4,Myc-Ub-K6 O,and Myc-Ub-K63O expression plasmids and treating with 50 μM MG132 for 6 h or 12 h,investigating K6 and K63-linked GATA4 ubiquitination and protein level.Performing in vitro ubiquitination assay using GATA4,DEC1,PARKIN,and Myc-Ub obtained by in vitro transcription and translation,examining the effect of DEC1 and PARKIN on GATA4 ubiquitination level.Results: Gata4 m RNA levels were not affected by Dec1 overexpression or knockdown in PE-treated NRVM and H9C2 cells.Dec1 overexpression could inhibit p-GATA4Ser105 and total GATA4 protein level in PE-treated NRVM and H9C2 cells.DEC1 could inhibit the upregulation of GATA4 in H9C2 cell nucleus and cytoplasm after PE stimulation,but didn’t change relative nucleoplasmic distribution.MG132,but not CQ,significantly inhibited DEC1-mediated GATA4 protein degradation.DEC1 promoted GATA4 K6 and K63-linked ubiquitination level.K6-linked ubiquitination,but not K63-linked ubiquitination,mediated GATA4 degradation via ubiquitin-proteasome pathway.DEC1 didn’t function as an E3 ligase to directly promote GATA4 ubiquitination.Conclusions: DEC1 promoted GATA4 K6-linked ubiquitination and degradation through ubiquitin-proteasome pathway.DEC1 was not an E3 ligase.Part Ⅳ Exploration of E3 Ligases in the Course of DEC1-mediated GATA4 Protein DegradationObject: To screen candidate molecules of E3 ligase in the process of DEC1-mediated GATA4 protein degradation by interacting protein mass spectrometry and explore the role of these candidate molecules on GATA4 ubiquitination and protein level as well as on pathological cardiomyocyte hypertrophy.Methods: Adenovirus overexpressing Gata4(AdGata4)was constructed,and H9C2 cells were infected with AdDec1 and AdGata4,respectively.After PBS treatment for 24 h,H9C2 cells were collected for co-IP experiment.DEC1 and GATA4 antibodies were used to enrich interacting proteins binding to DEC1 and GATA4,respectively,the enriched DEC1 and GATA4 interacting proteins were detected by mass spectrometry.E3 ligases binding to DEC1 and GATA4 proteins were selected as candidate E3 ligases.Expression plasmids of candidate E3 ligases were constructed,and these expression plasmids were transfected into 293 T cells with DEC1 and GATA4 expression plasmids,respectively.The binding of these candidate E3 ligases with DEC1 and GATA4 proteins was verified by co-IP assay.293 T cells were transfected with candidate E3 ligases expression plasmids,GATA4 expression plasmids and ubiquitin expression plasmids,respectively.DEC1 expression plasmids were used as positive control and co-IP assay was used to screen E3 ligase promoting GATA4 protein ubiquitination.In vitro ubiquitination assay was used to determine whether E3 ligase PRP19 could directly promote GATA4 protein ubiquitination.Adenovirus overexpressing Prp19(AdPrp19)was constructed,and H9C2 cells and NRVM cells were infected with AdPrp19,H9C2 and NRVM cells were stimulated with PE and treated with MG132.Western Blotting assay was performed to detect GATA4 protein expression levels in H9C2 and NRVM cells.H9C2 and NRVM cells infected with AdGata4 and/or AdPrp19 and stimulated with PE for corresponding time,were used to detect the effects of Gata4 and/or Prp19 overexpression on pathological cardiomyocyte hypertrophy.Results: The E3 ligases binding to DEC1 and GATA4 proteins were enriched by DEC1 and GATA4 antibody,respectively,the enriched protein complexes were analyzed by interacting protein mass spectrometry technique.Ten highly credible E3 ligases(BUB3,CORO1 C,CORO2B,EML4,FUS,GNB2,PRP19,SEC31 A,TAF3,UPF1)were screened out by combined analysis of mass spectrometry data.co-IP experiment confirmed that the ten E3 ligases had strong interaction with DEC1 and GATA4 proteins.In vivo and in vitro ubiquitination experiments showed that PRP19 was a direct E3 ligase that promoted GATA4 ubiquitination.Cell experiments showed that PRP19 could promote GATA4 degradation via ubiquitin-proteasome pathway in H9C2 and NRVM cells during pathological hypertrophy.NRVM cell rescue experiment results showed that overexpression of Prp19 could not only inhibit the NRVM pathological hypertrophy aggravated by Gata4 function enhancement,but also inhibit the up-regulation of Nppa and Nppb expression in NRVM and H9C2 cells stimulated by PE after Gata4 overexpression.Conclusions: The E3 ligase PRP19 could promote ubiquitination and degradation of GATA4 protein via ubiquitin-proteasome pathway,PRP19 could inhibit the pathological cardiomyocyte hypertrophy aggravated by enhanced GATA4 function.Part Ⅴ Identification of Lysine Sites for DEC1-PRP19-induced GATA4 Ubiquitination and DegradationObjective: To preliminarily identify DEC1-PRP19-mediated lysine site of GATA4 ubiquitination by consulting GATA4 protein amino acid sequence and ubiquitination experiment.Methods: Searching the literature about GATA4 protein research and inquiring the amino acid sequence of GATA4 protein using Uniprot protein database to obtain lysine sites in GATA4 protein.Mutating 11 lysine sites of GATA4 protein into glycine sites without ubiquitination modification function and constructing GATA4 expression plasmids containing intact single lysine site.These plasmids were transfected into 293 T cells with DEC1,PRP19 and ubiquitination plasmids,respectively.DEC1 and PRP19-promoted GATA4 ubiquitination lysine sites were detected using co-IP assay and Western Blotting assay.HA-GATA4-K256 O plasmids and Myc-Ub plasmids were transfected into 293 T cells.293 T cells were treated using 50 μM MG132 for corresponding time(6 h and 12 h).The expression level and ubiquitination level of GATA4 protein after MG132 treatment were detected by co-IP assay and Western Blotting assay.Results: Lysine 256 of GATA4 protein was identified to be the GATA4 ubiquitination lysine site by DEC1 and PRP19.Ubiquitination modification of GATA4 lysine 256 could mediate the degradation of GATA4 protein through ubiquitin-proteasome pathway.Conclusions: DEC1-PRP19 could promote ubiquitination modification of lysine 256 in GATA4 protein and mediate its degradation via ubiquitin-proteasome pathway.