Systematic C-degron activity profiling implicates conditional regulation of the CTLH E3 ligase complex in ribosome maturation.
Many E3 ubiquitin ligases recognise specific degron motifs located at protein termini, yet the physiological relevance of these N-degron and C-degron pathways remains poorly understood due to the limited number of validated substrates. Thus, the major aim of this thesis was to define how C-terminal degrons contribute to the regulation of protein stability in human cells and to determine the biological contexts in which they operate. To this end, I employed a Global Protein Stability (GPS) screening approach in combination with ORFeome expression libraries to assess the effect of C-terminal “capping” on the stability of thousands of full-length human proteins. This strategy enabled the systematic identification of proteins whose degradation is mediated by Cterminal degrons, providing the first proteome-wide genetic tool for exploring C-degron biology in the context of full-length substrates. This analysis identified that ZMYND19 harbours a C-degron targeted by the Muskelin substrate adapter of the CTLH E3 ligase complex. Characterisation of ZMYND19 stability revealed conditional regulation, whereby CTLH-mediated degradation was suppressed by TNF-a stimulation and enhanced by inhibition of mTOR signalling. These findings established that CTLH activity is not constitutive but instead subject to dynamic modulation from environmental cues. Parallel genetic and proteomic analyses identified two additional substrates of the CTLHMuskelin pathway, AAMP and AEN. I showed that the hitherto uncharacterized gene AAMP plays an essential role in ribosome biogenesis: like its orthologue in yeast, Sqt1, AAMP exhibits chaperone activity towards the ribosomal protein uL16 to enable ribosomal maturation. Altogether, these data define a novel C-degron pathway through which the Muskelin substrate adaptor connects conditional regulation of the CTLH E3 ligase complex to control of ribosome biogenesis.
Proteostasis, Protein Degradation, E3 Ligase, CTLH, C-degron
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