Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism

• Published in: Molecular cell Vol. 84; no. 10; pp. 1948 - 1963.e11
• Main Authors: Gottemukkala, Karthik V., Chrustowicz, Jakub, Sherpa, Dawafuti, Sepic, Sara, Vu, Duc Tung, Karayel, Özge, Papadopoulou, Eleftheria C., Gross, Annette, Schorpp, Kenji, von Gronau, Susanne, Hadian, Kamyar, Murray, Peter J., Mann, Matthias, Schulman, Brenda A., Alpi, Arno F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.05.2024
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; cryo-electron microscopy; Cryoelectron Microscopy; CTLH E3 ligase; cytotoxicity; degron mimicry; HEK293 Cells; Humans; internal degron; metabolic enzyme; metabolism; Nicotinamide-Nucleotide Adenylyltransferase - genetics; Nicotinamide-Nucleotide Adenylyltransferase - metabolism; NMNAT1; prodrug metabolism; Prodrugs - metabolism; Protein Binding; Substrate Specificity; ubiquitin proteasome system; ubiquitin-protein ligase; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; ubiquitylation; WDR26; yeasts; YPEL5; internal degron; WDR26; NMNAT1; ubiquitin proteasome system; CTLH E3 ligase; metabolic enzyme; prodrug metabolism; YPEL5; ubiquitylation; degron mimicry
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2024.04.014

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5’s N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism. [Display omitted] •Supramolecular WDR26-CTLH E3 complex ubiquitylates oligomeric metabolic enzyme NMNAT1•WDR26-CTLH E3 recognizes internal basic degron of NMNAT1 through WDR26 β-propellers•Degron mimicry of YPEL5’s N terminus inhibits NMNAT1 targeting by WDR26-CTLH E3•YPEL5-WDR26-CTLH E3 modulates cellular NMNAT1 amounts affecting its metabolic function Gottemukkala, Chrustowicz, Sherpa et al. reveal an intricate mechanism of selective binding and ubiquitylation of the oligomeric metabolic enzyme NMNAT1 by the supramolecular WDR26-CTLH E3 complex. Substrate targeting is inhibited by the CTLH E3 subunit YPEL5 mimicking substrate degrons, thus impacting the cellular abundance and metabolic function of NMNAT1.