Fig. 5: SIRT1 inhibits the AIF-CypA complex formation through deacetylating AIF-K295.

A The schematic representation of AIF illustrates its structural domains and binding motifs. The full-length AIF comprises a Mitochondrial Localization Sequence (MLS, depicted in green), a putative Transmembrane Domain (TM, also in green), a flavin adenine dinucleotide (FAD)-bipartite domain (colored orange), an NADH-binding motif (blue), and a C-terminal domain (red). The TM is bordered by two proteolytic cleavage sites: a mitochondrial processing peptidase (MPP) cleavage site at residue 54 and a calpain/cathepsin cleavage site at residue 102. Various proteins, including Hsp70, Cyclophilin A (CypA), phosphorylated H2AX (γH2AX), and Macrophage Migration Inhibitory Factor (MIF), interact with AIF at distinct regions. The numerical annotations correspond to specific amino acid positions. Additionally, the SIRT1-binding motif on AIF is located between residues 262 and 480. B, C GFP-tagged CypA was co-transfected with AIF-Flag or AIF-Flag K295R expression plasmids, followed by treatment with MNNG (12 h) or OGD (12 h). For MNNG treatment, cells were treated with 500 µM MNNG for 20 min, and then cells were maintained in fresh medium for another 12 h. Cells lysates were immunoprecipitated with FLAG-M2 beads followed by western blotting with anti-GFP and anti-AIF antibodies. D Binding of GST-AIF and GST-AIF-mutants to His-CypA in vitro. Bacterially purified GST, GST-AIF, and its mutants were incubated with His-CypA isolated from E. coli, and the GST pull-down assays were performed, followed by anti-GST and anti-His western blotting analysis. E 293 T cells co-transfected with the indicated plasmids were treated with or without MNNG (12 h) or OGD (12 h). The cells were then lysed and immunoprecipitated with FLAG-M2 beads, and the co-precipitated CypA was blotted for GFP antibody. F Control shRNA (shN) and SIRT1-specific shRNA stable-expressing (shR) SK-N-SH cells were treated with 500 µM MNNG for 20 min. The cells were maintained in fresh medium for another 12 h. After that, the cells were lysed and immunoprecipitated using an anti-AIF antibody (AIF IP), and co-precipitated proteins were blotted with anti-CypA and anti-SIRT1 antibodies (upper panel). CypA and SIRT1 expression levels were examined by western blot (lower panel). G SK-N-SH AIF stable knockdown (shR) cells rescued with Flag-AIF (WT, K295R/Q) were treated with or without MNNG as indicated. For MNNG treatment, cells were treated with 500 µM MNNG for 20 min, after which they were maintained in fresh medium for another 12 h. Subsequently, the cells were co-stained with anti-Flag antibody (green), DAPI (blue), and visualized using a fluorescence microscope (scale bars indicate 10 µm). H SK-N-SH AIF stable knockdown (shR) cells rescued with Flag-AIF (WT, K295R/Q) used in (G) were treated with or without MNNG as described above. The mitochondrial, cytosolic, and nuclear extracts were analyzed by western blotting for the presence of AIF. COX IV (mitochondrial marker), α-tubulin (cytosolic marker) and histone H3 (the nuclear marker) were used to control fractionation quality and protein loading. I SK-N-SH cells were transfected with either SIRT1 or the SIRT1-H363A mutant, as specified. Subsequently, cells were treated with 500 µM MNNG for 20 min, then cells were maintained in fresh medium for another 9 h. Following treatment, cell lysates were prepared and subjected to immunoprecipitation using an AIF antibody. The co-precipitated proteins, CypA and γH2AX, were analyzed by western blot. J SK-N-SH cells were transfected with GFP-CypA, AIF-Flag, and AIF-Flag K295R, as indicated. Cells were treated with 500 µM MNNG for 20 min, then cells were maintained in fresh medium for another 9 h, after which they were fixed and stained for Flag (red), GFP-CypA (green), and γH2AX (magenta). Confocal fluorescence microscopy was employed to visualize the stained cells. The nuclei were counterstained with DAPI (blue). Scale bars indicate 2 µm. Magnification scale bars indicate 0.25 µm. K SK-N-SH cells were transfected with GFP-CypA, AIF-Flag, and AIF-Flag K295R, as specified. Cells were treated with 500 µM MNNG for 20 min, then cells were maintained in fresh medium for another 9 h, then the cells were harvested and subjected to immunoprecipitation using GFP Magnetic Beads. The co-precipitated proteins were analyzed by western blotting with anti-Flag and anti-γH2AX antibodies (upper panel).