A chemical probe to modulate human GID4 Pro/N-degron interactions.

Area of Science:

• Molecular Biology
• Biochemistry
• Proteomics

Background:

• The C-terminal to LisH (CTLH) complex is a ubiquitin ligase involved in protein degradation.
• Its substrate receptor, Glucose-Induced Degradation 4 (GID4), recognizes proteins with Pro/N-degrons.
• The specific functions and substrates of the human CTLH complex are not well understood.

Purpose of the Study:

• To elucidate the function and identify substrates of the human CTLH complex.
• To characterize the role of Glucose-Induced Degradation 4 (GID4) in substrate recognition and regulation.
• To develop chemical tools for investigating CTLH complex biology.

Main Methods:

• Development and application of PFI-7, a selective chemical probe for human GID4.
• Proximity-dependent biotinylation coupled with quantitative proteomics to identify GID4 interactors.
• Analysis of GID4-regulated proteins to understand cellular level control.

Main Results:

• PFI-7 effectively antagonizes Pro/N-degron binding to human GID4.
• Identified nucleolar proteins, including RNA helicases DDX21 and DDX50, as GID4 interactors.
• Discovered that GID4 regulates the cellular levels of proteins like HMGCS1, a metabolic enzyme, via degradative and non-degradative mechanisms.

Conclusions:

• Human GID4 targets Pro/N-degron proteins through both degradation and non-degradation pathways.
• PFI-7 is a valuable tool for studying CTLH complex function and developing targeted protein degradation strategies.
• The findings expand our understanding of ubiquitin ligase complexes and their roles in cellular regulation.