Dissecting substrate recognition by GID Ubiquitin ligases
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Abstract
Protein degradation is an essential process for maintaining the health of the cell, with a variety of different functions in the maintenance of homeostasis, regulation of processes in the cell, the response to environmental stresses, and quality control of the proteome. Perturbation of this system leads to a variety of diseases, including neurodegenerative diseases such as Alzheimer's disease or frontotemporal dementia.
To degrade proteins, the cell uses two main pathways, autophagy and the ubiquitin proteasome system, the latter of which will be the focus of this study. In this pathway, ubiquitin is attached to substrate proteins by the action of E3 ligases. One such E3 ligase of rising importance is the GID/CTLH complex, which was originally found to be involved in the degradation of gluconeogenic enzymes starting with proline via its substrate receptor Gid4. In more recent years, further substrate receptor proteins have been discovered for the GID complex with Gid10 and Gid11, together with the complex’s ability to non-canonically recognize substrates via WDR26 in human cells.
Thus far, Gid11 is relatively poorly understood, besides it seeming to recognize N-terminal threonine residues. One of the aims of this study is to better characterize Gid11, analyze its substrate specificities, and identify residues or regions within Gid11 important for substrate recognition. For this purpose, this study the tandem-fluorescent timer approach was used. This study highlights the importance of the third intrinsically disordered region of Gid11, as well as a variety of residues within its binding pocket. Furthermore, it shows that N-termini recognized by Gid11 are usually non-acetylated. Additionally, the Gid11-dependent turnover of two proteins without threonine N-degrons was investigated, leading to the hypothesis that Blm10 is potentially recognized via the fifth intrinsically disordered region of Gid11. A potential example of trans-ubiquitination of Cpa1 via recognition of its complex partner Cpa2 was investigated and disproven.
There are several more proteins such as Hsm3 whose turnover depends on the GID complex, but which do currently not possess a known substrate receptor. Using overexpression of potential substrate receptors to compete for the binding of Gid5, it could be shown that this approach is a viable strategy to find additional, currently unknown Gid5-dependent substrate receptor proteins of the GID complex.
Additional experiments were conducted to investigate the functional conservation of various GID components, both in different yeast species in case of Gid11, and in human cells in the case of core GID components, showing that most human homologs of GID components are non-functional in yeast.
As a side project, the specificity of human ATE1, NTAN1, and NTAQ1 was investigated, highlighting differences between these enzymes and their human counterparts.
Overall, this study provides a characterization of Gid11, its substrate specificities, and identifies several key features necessary for its function. Furthermore, it highlights potential ways to identify further, currently unknown substrate receptors of the GID complex.