Journal: bioRxiv

Article Title: Proteome-wide C-degron activity profiling connects conditional regulation of the CTLH E3 ligase complex to ribosome biogenesis

doi: 10.64898/2026.01.14.698769

Figure Lengend Snippet: (A-B) Cell-to-cell variability in ZMYND19 stability. (A) ZMYND19 expressed in the context of the GPS system escapes degradation in a variable proportion of the cells. (B) Following isolation of the GFP bright cells by FACS, the population quickly reverted towards the GFP dim state. (C) A chemical screen to identify conditions which impact ZMYND19 degradation. HEK-293T cells expressing ZMYND19 in the context of the GPS vector were subjected to the indicated treatments and the effect on ZMYND19 degradation assessed by flow cytometry. (D-E) Inhibition of mTOR signaling potentiates ZMYND19 degradation. (D) Overnight treatment with the mTOR inhibitor Torin-1 abolishes phosphorylation of p70 S6 kinase. (E) Amino acid withdrawal or Torin-1 treatment enhances ZMYND19 degradation, overriding the effects of TNF-α stimulation. See also . (F) Conditional regulation of the CTLH GID4 substrate HMGCS1. HMGCS1 is more abundant following TNF-α stimulation and less abundant following Torin-1 treatment, as assessed by immunoblot. (G) The association between ZMYND19 and Muskelin is maintained in GFP bright cells where GPS-ZMYND19 escapes degradation. Following overnight TNF-α stimulation, cells expressing GPS-ZMYND19 were partitioned into GFP dim and GFP bright populations by FACS and the interaction between ZMYND19 and Muskelin assessed via co-immunoprecipitation. (H) Effect of TNF-α stimulation and mTOR inhibition on the abundance of CTLH subunits, as assessed by immunoblot. (I-J) Differential requirements for RANBP9 and RANBP10 for CTLH-mediated degradation of ZMYND19 upon mTOR inhibition. Using sgRNAs that result in efficient gene disruption as assessed by immunoblot (I) , the effects of targeting RANBP9 and RANBP10 either individually or simultaneously on ZMYND19 degradation were assessed by flow cytometry (J) . Ablation of RANBP9 prevents ZMYND19 degradation at steady state; upon mTOR inhibition, however, targeting of RANBP9 has little impact on ZMYND19 stability, and dual ablation of RANBP9 and RANBP10 is necessary before abrogated degradation is observed.

Article Snippet: Primary antibodies used were: rabbit α-MAEA (Proteintech, #28363-1-AP), rabbit α-TWA1 (Novus Biologicals, #NBP1-32596), mouse α-Muskelin (Santa Cruz, #sc-398956), mouse α-RanBP9 (Santa Cruz, #sc-271727), rabbit α-RanBP10 (Proteintech, #21107-1-AP), mouse α-ARMC8 (Santa Cruz, #sc-365307), rabbit α-WDR26 (Bethyl, #A302-244A), rabbit α-YPEL5 (Proteintech, #11730-1-AP), rabbit α-AAMP (Abcam, #EPR12369), rabbit α-ISG20L2 (Proteintech, #24639-1-AP), rabbit α-HMGCS1 (Proteintech, #17643-1-AP), mouse α-UBE2H (Santa Cruz, #sc-100620), rabbit α-uL16/RPL10 (Proteintech, #17013-1-AP), rabbit α-uL14/RPL23 (Abcam, #ab264369), mouse α-eS6/RPS6 (Cell Signaling Technology, #2317), mouse α-β-actin (Merck, #A2228), mouse α-vinculin (Merck, #V9131), rabbit α-HA-tag (Cell Signaling Technology, #C29F4), mouse α-FLAG (Merck #F1804), rabbit α-GFP (Abcam, #ab290), mouse α-GFP (Roche, #11814460001) and rabbit α-Phospho-p70 S6 Kinase (Thr389) (Cell Signaling Technology, #9205).

Techniques: Isolation, Expressing, Plasmid Preparation, Flow Cytometry, Inhibition, Phospho-proteomics, Western Blot, Immunoprecipitation, Disruption

Journal: bioRxiv

Article Title: Proteome-wide C-degron activity profiling connects conditional regulation of the CTLH E3 ligase complex to ribosome biogenesis

doi: 10.64898/2026.01.14.698769

Figure Lengend Snippet: (A) Schematic representation of the stability profiling experiment, in which the Ultimate ORFeome GPS expression library was used to identify proteins exhibiting increased stability in MAEA KO and TWA1 KO cells. (B-C) Identification of AAMP, AEN and NATD1 as CTLH substrates. Screen profiles are depicted in (B) , while individual validation experiments by flow cytometry in the presence and absence of Torin-1 (dotted lines) are shown in (C) . See also . (D) Schematic representation of the proteomic experiment, in which TMT mass spectrometry was used to identify proteins more abundant in cells lacking the CTLH subunits TWA1 and Muskelin. Cells expressing GPS-ZMYND19 were used to allow purification of populations of cells in which TWA1 and Muskelin had been successfully ablated by FACS. See also . (E-F) AAMP is more abundant in cells lacking CTLH subunits. The scatterplot in (E) represents the mean fold change of each protein quantified in both TWA1 KO and Muskelin KO cells, with proteins showing a >1.2-fold increase in abundance in both conditions highlighted in red; the performance of AAMP compared to the known substrate HMGCS1 is depicted in (F) . Exogenous expression of ZMYND19 precludes assessment of the true magnitude of the stabilization of the endogenous ZMYND19 protein in this experiment.

Article Snippet: Primary antibodies used were: rabbit α-MAEA (Proteintech, #28363-1-AP), rabbit α-TWA1 (Novus Biologicals, #NBP1-32596), mouse α-Muskelin (Santa Cruz, #sc-398956), mouse α-RanBP9 (Santa Cruz, #sc-271727), rabbit α-RanBP10 (Proteintech, #21107-1-AP), mouse α-ARMC8 (Santa Cruz, #sc-365307), rabbit α-WDR26 (Bethyl, #A302-244A), rabbit α-YPEL5 (Proteintech, #11730-1-AP), rabbit α-AAMP (Abcam, #EPR12369), rabbit α-ISG20L2 (Proteintech, #24639-1-AP), rabbit α-HMGCS1 (Proteintech, #17643-1-AP), mouse α-UBE2H (Santa Cruz, #sc-100620), rabbit α-uL16/RPL10 (Proteintech, #17013-1-AP), rabbit α-uL14/RPL23 (Abcam, #ab264369), mouse α-eS6/RPS6 (Cell Signaling Technology, #2317), mouse α-β-actin (Merck, #A2228), mouse α-vinculin (Merck, #V9131), rabbit α-HA-tag (Cell Signaling Technology, #C29F4), mouse α-FLAG (Merck #F1804), rabbit α-GFP (Abcam, #ab290), mouse α-GFP (Roche, #11814460001) and rabbit α-Phospho-p70 S6 Kinase (Thr389) (Cell Signaling Technology, #9205).

Techniques: Expressing, Biomarker Discovery, Flow Cytometry, Mass Spectrometry, Purification