Fig 2: Diphthamide promotes TORC1 and mTORC1 signaling. (A) Diphthamide promotes Sch9 phosphorylation in yeast. A chemical cleavage assay was used to detect the phosphorylation level of Sch9. Endogenous Sch9 was tagged with triple Flag tag (3xFlag) at the C-terminal using homologous recombination to allow detection of Sch9 by Flag antibodies. In the WT strain, C-terminal of Sch9 was shown as multiple higher molecular weight bands after chemical cleavage, indicating a higher phosphorylation level of Sch9 in the WT strain. (B) Diphthamide promotes 4EBP1 phosphorylation in HEK293T cells. Dph4 was knocked out using CRISPR. The phosphorylation level of 4EBP1 was detected by Western blot. (C) Diphthamide promotes 4EBP1 phosphorylation in HEK293T cells. Either a control vector or Dph4 encoding construct was reintroduced into HEK293T Dph4 KO cells. The phosphorylation level of 4EBP1 was detected by Western blot. (D) Diphthamide level decreases under rapamycin treatment. HEK293T cells were treated with 0, 10, or 100 nM rapamycin, and the level of diphthamide modification was detected with a diphtheria toxin-mediated ADP-ribosylation reaction with a fluorescent-labeled NAD+ analog. The level of EF2 protein was detected using Western blot. (E) Diphthamide level increases with amino acid supplement. HEK293T cells were cultured without amino acid or with amino acid. The level of diphthamide modification was detected with a diphtheria toxin-mediated ADP-ribosylation reaction with a fluorescent-labeled NAD+ analog. The level of EF2 protein was detected using Western blot.

Fig 3: WB analysis of mTOR pathway proteins in mouse and human brains. (A) Protein was analyzed by WB. Gba-/-(NestinCre) mice brains (Gba-/-) (29 days) were compared with WT mice (35 days). Human GD2 brain (15 months) and GD3 hippocampus (11.5 years) were compared with healthy control hippocampus (12 years). (B) Fold change of WT level after normalization by ß-actin. Phospho-S6-RP (arrow)/S6-RP, phospho-4E-BP1/4E-BP1, and phospho-mTOR were increased in Gba-/-(NestinCre) mice brains compared with WT mice. Student’s t-test (replicate assays, n = 2 mice brains). In human samples, phospho-S6-RP/S6-RP, phospho-4E-BP1/4E-BP1, and mTOR were increased in GD2 brain and GD3 hippocampus compared with healthy control hippocampus. Phospho-mTOR was undetectable (U.D.) in human brains (replicate experiments, n = 1 human brain). Student’s t-test (* p < 0.05; ** p < 0.01). ns, not statistically significant.

Fig 4: The effect of 4E-BP(4Ala) on translation of reporter mRNAs and on YB-1 synthesis in HeLa cells.(A) 105 HeLa cells were transfected by 1 or 2 µg pcDNA3-3HA-4E-BP1(4Ala) or 2 µg pcDNA3-HA plasmids, cultivated for 36 h and used for Western-blot analysis with anti-4E-BP1 antibody. (B) 4E-BP1(4Ala)-overexpressing or control HeLa cells (105 each) were transfected by reporter Firefly luciferase mRNAs with YB-1 mRNA- or globin mRNA 5' UTR and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase (FLuc and RLuc, respectively). The FLuc/RLuc ratio for the control (untreated cells) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. (C) 4E-BP1(4Ala)-overexpressing or control HeLa cells (106 each) were labeled with [35S]-methionine for 2 h, harvested and lysed. Cell lysates were used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [35S]-labeled proteins were detected by autoradiography. (D) Biotinylated, capped luciferase mRNA with YB-1 mRNA 5' UTR (0.32 pmol) was incubated in 150 µl lysates of 4EBP(4Ala)-overxpressing HeLa cells (2 × 105) or control HeLa cells (2 × 105) and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE and analyzed by Western blotting.

Fig 5: Cck1R activation normalizes mTORC1 signaling activity and restores cerebellar marker Calb1 in ATXN2[Q127] mice(A) Phosphorylation of the ribosomal protein S6 (pS6) and eukaryotic translation initiation factor 4E binding protein 1 (p4e-bp1) is increased and the cerebellar marker Calb1 is decreased in the cerebellum of ATXN2[127] mice (13 weeks of age) compared to WT mice. Administration of the Cck1R-selective agonist A71623 to ATXN2[127Q] mice for 24 h improves cerebellar phosphorylation of S6 and 4e-bp1 and increases levels of Calb1. Each lane represents extract from an individual mouse. Actb is used as a loading control, and blots are from replicate experiments.(B) Quantification of pS6, p4e-bp1, and Calb1 is shown.Data are mean ± SD. ns, p > 0.05; *p < 0.05; **p < 0.01; and ***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparisons test.