Journal: bioRxiv

Article Title: Cerebral Small Vessel Disease genetic determinant TRIM47 controls brain homeostasis via the NRF2 antioxidant system

doi: 10.1101/2024.10.08.616723

Figure Lengend Snippet: a-b. qPCR analysis of ( a ) NQO1 and ( b ) HMOX1 gene expression in siCont or siNRF2 treated HBMEC and co-transduced with a control or TRIM47 lentivirus for 24h. Data were normalized to cyclophilin (n=3 experiments). * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001, One-way ANOVA. c. HO1 promoter luciferase reporter assay. TRIM47 cDNA expression plasmid (TRIM47) or empty expression plasmid (pcDNA) were co-transfected with HO1 promoter-luciferase constructs (HO1-4.5bp, HO1-4.5bp with 2 or 3 mutations) or a pGL3 empty vector in HeLa, and luciferase activity was measured. Values represent the fold change in relative luciferase activity over pGL3 vector (n=4 experiments). * P<0.05; ** P<0.01; **** P<0.0001, One-way ANOVA; ### P<0.001: compared to pGL3 + Lenti control condition, Student’s t-test. d. TRIM47 and KEAP1 interaction was assessed by Co-IP assay in whole cell lysates from HEK293. Cells were transfected with TRIM47-myc pcDNA, KEAP1-Flag or both expression plasmids. Lysates were immunoprecipitated with myc antibody and immuno-blotted for myc (TRIM47) and KEAP1. e. Immunoblot and quantification of WB for NRF2 and KEAP1 in control and TRIM47-deficient HBMEC treated with DMSO or MG132 for 5h (n=4 experiments). Data were normalized to α-tubulin.* P<0.05; ** P<0.01, Student’s t-test. f-g. qPCR analysis of ( f ) NQO1 and ( g ) HMOX1 gene expression in HBMEC treated with control, TRIM47 or NRF2 siRNA for 72h and with tBHQ (10µM) for 24h (n=3 experiments). ** P<0.01; *** P<0.001; **** P<0.0001, One-way ANOVA; ##, P<0.01 Student’s t - test. All graphical data are mean ± s.e.m. h. Model of TRIM47 activation of the antioxidant NRF2 pathway in endothelial cells. 1. In basal conditions, TRIM47 binds to KEAP1 in the cytoplasm and thus alleviates KEAP1-dependent NRF2 degradation and increases NRF2 protein stabilization that can translocate to the nucleus to drive the expression of NRF2-target genes ( HMOX1 , NQO1 ) promoting cellular antioxidant protection. 2. In the absence of TRIM47 (TRIM47 siRNA condition), KEAP1 strongly binds to NRF2 leading to its ubiquitination and degradation by proteosomal complex, thus leading to the decreased of the protective NRF2 pathway.

Article Snippet: Flag-Keap1 was a gift from Qing Zhong (Addgene plasmid #28023; http://n2t.net/addgene:28023 ; RRID:Addgene_28023). pCDNA3-Myc3-Nrf2 was a gift from Yue Xiong (Addgene plasmid #21555; http://n2t.net/addgene:21555 ; RRID:Addgene_21555). pHOGL3/4.5 and pHOGL3/4.5 Triple mutant plasmids were a gift from Prof. Anupam Agarwal (University of Alabama at Birmingham, US).

Techniques: Expressing, Transduction, Control, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Construct, Activity Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Activation Assay