Fig 2: PRAS40 98-110 is sufficient but not required for PRAS40–P65 association. (a) The PRAS40 structure was predicted by the I-TASSER algorithm and visualized using the PyMOL software (Left). The truncations of PRAS40 were produced based on the predicted structure of PRAS40 (Right). ? means could interact with P65; × means could not interact with P65 according to the results of a GST co-precipitation assay (b, c). (b) Truncations of PRAS40 with GST tag protein were co-transfected withVenus-P65 (V-P65, P65 protein with the Venus-Flag tag). The GST, Venus and truncations of PRAS40 (1-23, 1-51,1-75 and 110-256) could not co-precipitate P65 (lanes 1–3). However, the full length of PRAS40 and the truncations, including 1-110, 1-125, 1-170, 75-256, 98-256, 23-256 and 44-256 of PRAS40, could co-precipitate P65. 14-3-3?was used as the positive control for PRAS40 binding in this experiment. (c) The small truncation 98-110 of PRAS40 could co-precipitate P65 (lane 2 vs lane 1). But it could not significantly affect the co-precipitation of P65 by full-length PRAS40 (lane 6 vs lane 5). Lanes 3 and 4 were negative controls. (d) The 98-100 truncation of PRAS40 did not significantly regulate NF-?B transcriptional activity with or without tumor necrosis factor-a (TNF-a) stimulation (20 ng/ml, 2 h). (e) The proteins in the 3?B-Luc group cells were blotted to validate the transfection.

Fig 3: p65 180-200 peptide disrupts the PRAS40–P65 interaction and regulates NF-?B transcriptional activity. (a) The Venus-tagged p65 180-200 peptide could disrupt the PRAS40–P65 association (lane 4 vs lane 3). There is no significant difference between the cells co-transfected with GST PRAS40 DNA and 0.75 or 1 µg of Venus-P65 (lane 2 vs lane 3). (b) HA-tagged p65 180-200 could disrupt the PRAS40 association with exogenously expressed or endogenous p65 (lane 1 vs lane 3; lane 2 vs lane 5). The remaining blots served as negative controls. The blot of the Ex-/En-P65 shows the exogenous and endogenous P65 expression, respectively. (c) In all, 46.3% of NF-?B transcriptional activity in the 3?B-Luc group cells with PRAS40 overexpression was reduced by the 180-200 fragment of p65 (P<0.001). The mutants of PRAS40 increased the NF-?B transcription activity comparing to the pcDNA control group. There are no significant differences of relative activity between the groups of ConA-Luc. NS=P>0.05, *P<0.01. (d) The p65 180-200 peptide significantly reduced the basal NF-?B transcription activity in cells with endogenous PRAS40 expression. *P<0.01. (e) The 3?B-Luc group cells with Venus and Venus-P65 180-200 used in the above experiment (d) were utilized to validate the transfection efficiency. Flag was used as the referral control of transfection because flag tag was included in the Venus tag vector in this study.

Fig 4: Endothelial-specific PRAS40 deficiency promotes atherogenic remodeling in vivo. (a) Schematic diagram of the partial carotid ligation. Arrows indicate direction of blood flow. Red dotted lines represent cross sections for quantitative analyses at indicated predefined distances from the proximal end of the vessel. ECA, external carotid artery; ICA, internal carotid artery; STA, superior thyroid artery; OA, occipital artery (b) Mice with tamoxifen-induced endothelium-specific PRAS40 deficiency (EC-PRAS40-KO) and tamoxifen-treated Cre-negative littermates (WT controls) were continuously exposed to western diet for a total of 10 weeks. Four weeks after partial ligation, left common carotid arteries as well as sham-operated right common carotid arteries were harvested for further analyses. Shown are representative carotid cross sections at five predefined distances from the proximal end of the carotid artery (0 µm, 1000 µm, 2000 µm, 3000 µm, 4000 µm) stained with elastic stain. Scale bars in black 100 µm. (c) Mean intimal cross sectional areas (including plaque and neointima) were quantified at each predefined distance (based on analysis of 4 different animals per genotype). Shown are absolute plaque areas as well as plaque areas in relation to the contralateral sham operated vessel size (area enclosed by external elastic lamina). Data represent mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed student’s t-test).

Fig 5: AIF deficiency stimulates Akt and mTOR activity. (A–B) Immunoblot analyses were performed on (A) muscle and (B) cerebellar tissues from 3 to 6 month-old animals. Densitometry is relative to wt littermates and reported as mean ± SEM. Antibodies against total and phosphorylated forms of Akt, 40-kDa proline-rich Akt substrate (PRAS40), p70 S6 kinase (P70-S6K), ribosomal Protein S6 (RPS6) were used (n = 4–9 per genotype). GAPDH and actin were used as loading control (***p < 0.001, **p < 0.01, *p < 0.05). Color code is: black = wt (for KI); red = KI; dark grey = wt (for Hq); green = Hq. (C) Immunoblot analyses on muscle and cerebellar tissue from Hq mice and littermate controls following 2 weeks of rapamycin or vehicle treatment. Compared to vehicle-treated mice, animals of the rapamycin-group showed reduced mTOR activity, as revealed by decreased pRPS6. (D) Grip strength test was performed in wt and Hq mice injected with vehicle or rapamycin. Compared to mock treatment, rapamycin improved muscular performance in Hq animals (n = 9–10 per group). (E) RT-PCR analysis of Mthfd2 expression in wt and Hq mice treated with rapamycin. Compared to vehicle-treated animals, rapamycin-injected mice displayed a significant decrease in Mthfd2 mRNA expression within the quadriceps muscle and cerebellum (n = 5–6 per group). (F) Schematic comparison of molecular and phenotypic defects in AIF deficient animals. Compared to Harlequin mutant mice, Aifm1 (R200 del) knockin animals have a more homogeneous phenotype, with common pathological traits with AIFM1-linked human patients, such as COX defects associated with early-onset myopathy. In Aifm1 (R200 del) knockin animals, molecular defects include CI and CIV impairment, altered carbon metabolism and aberrant Akt/mTOR activity.