Identification and characterization of WDR26-dependent hGID substrates
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Date
2024
Publication Type
Doctoral Thesis
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yes
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Abstract
Reversible post-translational modifications (PTMs) influence multiple signaling pathways by altering protein degradation, localization, interactions, and signaling dynamics. One of the most prevalent PTMs is Ubiquitin (Ub), a highly conserved 76-amino-acid protein that is covalently attached to lysine residues of proteins, where the final enzymatic component of this process is conducted by ubiquitin E3 ligases. Ubiquitination of such targets is firmly established to regulate multiple cellular functions, including cell cycle progression, DNA repair, and signal transduction by specific targeting of substrates in a spatiotemporal format via diverse substrate adaptors. One recently emerging scenario regards nutrient signaling, where superfluous gluconeogenic enzymes are targeted for degradation by the yeast GID ligase, however, mammalian functions have diverged to target distinct substrates related to proliferation in degradative and nondegradative formats via multiple substrate adaptors.
In this study, we sought to characterize WDR26-dependent substrates of the human GID complex (hGID), where we demonstrated that the WDR26-dependent substrate, Hbp1, is targeted by RanBP9-WDR26 in a WD40 domain dependent manner. Furthermore, we biochemically characterized patient and WD40 mutations in WDR26, which displayed alterations in Hbp1 ubiquitination, binding dynamics, or association with hGID complex components. With the goal of identifying additional WDR26-dependent targets of hGID, we additionally employed a dual mass spectrometry approach using both AP-MS and SILAC diGly-MS remnant profiling to identify proteasomal targets of WDR26-hGID. Here, we identified UCK2 as a novel WDR26-dependent substrate, implicated in S phase cell cycle progression associated with WDR26-depletion. As UCK2 is a rate limiting enzyme in pyrimidine metabolism, this work substantiates the functionality of the hGID ligase in regulating rate limiting metabolic functions in mammalian cells, providing mechanistic insight as to how this molecular machine displays conserved essentiality in mammalian formats and potentially contributes to developmental disease progression in associated pathological scenarios.
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Examiner : Peter, Matthias
Examiner: Corn, Jacob
Examiner: Neurohr, Gabriel
Examiner : Beli, Petra
Examiner : Pritz, Stephan
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ETH Zurich
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Subject
Cell cycle regulation; E3 ligases; hGID/CTLH; Oligomerization; substrate receptors; Ubiquitin
Organisational unit
03595 - Peter, Matthias / Peter, Matthias