Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet: (A) Ablation of RBX1 diminishes amino acid-induced mTORC1 activation in EMSCs. Three gRNAs designed to target distinct exon regions of RBX1 were used. Cells were starved in amino acid-deprived medium for 50 min and treated with 1× amino acids for 10 min. gRNA targeting GFP was used as a control. Note that RBX1–3 gRNA failed to ablate RBX1. (B) Ablation of RBX1, but not RBX2, diminishes amino acid-induced mTORC1 activation in EMSCs. Cells were treated as in (A). Levels of phosphorylated S6K1 in the indicated cells (amino acid-replete conditions) were quantified and expressed as the ratio of pS6K/S6K1. * p < 0.05, ** p < 0.01, mean ± SD, n = 4. (C) Ablation of RBX1 diminishes CQ/MG-induced mTORC1 activation in Ragulator-deficient cells. RBX1 is ablated in p18/LAMTOR1 knockout (KO) EMSCs. Cells were treated with CQ (50 μM) or/and MG (20 μM) in amino acid-free medium for 45 min. (D) Ablation of RBX1 diminishes Rheb ubiquitination. RBX1 KO EMSCs were lysed under amino acid-replete conditions in RIPA buffer containing the deubiquitinase inhibitor (100 mM) N-ethylmaleimide (NEM). The lysates were subjected to SDS-PAGE, and levels of Rheb and Ub-Rheb were monitored with a Rheb antibody. Non-Ub-Rheb and Ub-Rheb are indicated. (E) MLN4924 specifically inhibits mTORC1 activity induced by amino acids in EMSCs. Cells were treated with the indicated concentrations of MLN4924 for 16 h. Cells were amino acid starved for 50 min and re-stimulated with 1× amino acids for 10 min. Levels of phosphorylated S6K1 in the indicated cells (amino acid-replete conditions) were quantified and expressed as the ratio of pS6K/S6K1. **** p < 0.0001, mean ± SD, n = 4. (F) MLN4924 inhibits mTORC1 activity induced by amino acids in both control and DEPDC5 KO HEK293T cells. Cells were treated with MLN4924 (1 μM) as in (E). Cells were amino acid starved for 50 min and re-stimulated with 1× amino acids for 10 min. The effect of DEPDC5 ablation was confirmed by the high levels of mTORC1 activity resistant to amino acid starvation. (G) MLN4924 diminishes levels of Ub-Rheb in HEK293T cells. Cells were treated with MLN4924 (1 μM) for 16 h and lysed as in (D), and the lysates were subjected to immunoblotting with a Rheb antibody.

Article Snippet: RBX2 polyclonal antibody , Proteintech , Cat# 11905-1-AP; RRID:AB_10697836.

Techniques: Activation Assay, Control, Knock-Out, Ubiquitin Proteomics, SDS Page, Activity Assay, Western Blot

Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet:

Article Snippet: RBX2 polyclonal antibody , Proteintech , Cat# 11905-1-AP; RRID:AB_10697836.

Techniques: Virus, Recombinant, Magnetic Beads, Control, Negative Control, Ubiquitin Proteomics, shRNA, Plasmid Preparation, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1

doi: 10.1101/2024.10.07.617049

Figure Lengend Snippet: a, Alignment of ASB8 and ASB9. ELOB/C (dark green) bound to ASB9 (dark teal) from 6V9H was aligned to ELOB/C (light green) bound to the ASB8 (pale cyan) in our KPT-185-XPO1•ASB8 DN16 •ELOB/C cryo-EM structure (0.809 Å r.m.s.d.). The α1 and α2 helices of ANK5-7 are labeled, and the SOCS boxes are marked with a dashed line. b, A model of KPT-185-XPO1•CRL5 ASB8 generated by a series of alignments. The cryo-EM structure of KPT-185-XPO1•ASB8•ELOB/C was first aligned with ELOB/C-CUL5 ntd (4JGH) (r.m.s.d 1.151 Å). The overlayed ELOB/C-CUL5 ntd was then aligned to CUL5 FL (7ONI, r.m.s.d. 0.843 Å) to position the full length CUL5 into the model. Finally, the four-helix bundle and α/β subdomains (CUL5 residues 401-687) of the overlayed CUL5 FL (7ONI) and the NEDD8-CUL5 ctd •RBX1 (3DQV) were aligned (r.m.s.d. 1.143 Å) to model in the NEDDylated CUL5 and the released/mobile RBX1 (copies from both asymmetric units shown). The mobile RBX1/2 is expected to allow the associated E2 enzyme to reach exposed lysine residues of XPO1.

Article Snippet: 18 h post-KPT-185 treatment, cells were lysed in RIPA buffer and immunoblotting was performed using primary antibodies: XPO1 (CRM1 rabbit polyclonal H-300; sc-5595, Santa Cruz or CRM1 mouse monoclonal (C-1): sc-74454), NAE1 (Cell Signaling Technology, #14321), ELOB (Proteintech, 12450-1-AP), ELOC (Bethyl Laboratories, A304-008A), CUL5 (Bethyl Laboratories, A302-173A), RBX1 (Cell Signaling Technology, #11922), RBX2 (Sigma Aldrich, HPA036995) and GAPDH (Loading Control Antibody Sampler Kit #5142, Cell Signaling Technology).

Techniques: Cryo-EM Sample Prep, Labeling, Generated

Journal: bioRxiv

Article Title: Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1

doi: 10.1101/2024.10.07.617049

Figure Lengend Snippet: a, Alignment of ASB8 and ASB9. ELOB/C (dark green) bound to ASB9 (dark teal) from 6V9H was aligned to ELOB/C (light green) bound to the ASB8 (pale cyan) in our KPT-185-XPO1•ASB8 DN16 •ELOB/C cryo-EM structure (0.809 Å r.m.s.d.). The α1 and α2 helices of ANK5-7 are labeled, and the SOCS boxes are marked with a dashed line. b, A model of KPT-185-XPO1•CRL5 ASB8 generated by a series of alignments. The cryo-EM structure of KPT-185-XPO1•ASB8•ELOB/C was first aligned with ELOB/C-CUL5 ntd (4JGH) (r.m.s.d 1.151 Å). The overlayed ELOB/C-CUL5 ntd was then aligned to CUL5 FL (7ONI, r.m.s.d. 0.843 Å) to position the full length CUL5 into the model. Finally, the four-helix bundle and α/β subdomains (CUL5 residues 401-687) of the overlayed CUL5 FL (7ONI) and the NEDD8-CUL5 ctd •RBX1 (3DQV) were aligned (r.m.s.d. 1.143 Å) to model in the NEDDylated CUL5 and the released/mobile RBX1 (copies from both asymmetric units shown). The mobile RBX1/2 is expected to allow the associated E2 enzyme to reach exposed lysine residues of XPO1.

Article Snippet: 18 h post-KPT-185 treatment, cells were lysed in RIPA buffer and immunoblotting was performed using primary antibodies: XPO1 (CRM1 rabbit polyclonal H-300; sc-5595, Santa Cruz or CRM1 mouse monoclonal (C-1): sc-74454), NAE1 (Cell Signaling Technology, #14321), ELOB (Proteintech, 12450-1-AP), ELOC (Bethyl Laboratories, A304-008A), CUL5 (Bethyl Laboratories, A302-173A), RBX1 (Cell Signaling Technology, #11922), RBX2 (Sigma Aldrich, HPA036995) and GAPDH (Loading Control Antibody Sampler Kit #5142, Cell Signaling Technology).

Techniques: Cryo-EM Sample Prep, Labeling, Generated