Just as your household has a recycling system for waste, cells have sophisticated mechanisms to break down old or damaged proteins to keep everything running smoothly. This system uses chemical tags called ubiquitin to mark proteins for disposal. Researchers from the Structural Genomics Consortium (SGC) have achieved a significant breakthrough with the development of PFI-7, a chemical probe that interferes with a key enzyme’s ability to attach these ubiquitin tags. By using PFI-7 to explore human Glucose-Induced Degradation 4 (GID4), an essential but understudied component of this disposal system, they have revealed new insights into the interactions of GID4. 

A Closer Look at the CTLH Complex

Let’s think of protein degradation as a vital recycling process in which proteins are broken down into their constituent, amino acids. This helps maintain cellular balance, regulate protein levels, and remove damaged proteins. One of the key mechanisms involved in protein degradation is the ubiquitin-proteasome system, where proteins are tagged with ubiquitin for degradation. Specific sequences within the proteins, called degrons, are recognized by E3 ubiquitin ligases, marking them for degradation. 

The ability to manipulate degron recognition pathways opens up possibilities for developing drugs that can precisely target and degrade pathogenic proteins, especially for diseases like cancer and neurodegenerative disorders, linked with the accumulation or overproduction of specific proteins. This approach, called “targeted protein degradation”, is particularly promising for conditions where traditional small-molecule drugs are ineffective or where protein accumulation plays a central role in disease pathology.

The C-terminal to LisH (CTLH) complex, an E3 ubiquitin ligase, targets proteins for degradation. GID4, a subunit of this complex, plays a critical role in recognizing N-terminal degrons, particularly those with proline residues. Despite its importance, the specific functions and substrates of the CTLH complex in humans have remained largely enigmatic. 

“Bioinformatic prediction has identified potential substrates based on Pro/N-degron sequences, showing interactions with the CTLH complex. However, a comprehensive assessment of GID4-dependent interactors and its role in human proteome regulation is still lacking”, explains Dr. Cheryl Arrowsmith, Chief Scientist at SGC at the University of Toronto, who supervised the effort.

Discovery of a potent ligand targeting GID4

To address this knowledge deficit and explore the E3 ligase activity of the CTLH complex, the researchers sought to develop a chemical probe. Their efforts focused on the evolutionarily conserved substrate receptor of CTLH, GID4. In partnership with Pfizer and following a successful initial hit-ID campaign and structure-based drug design, a novel chemical handle (PFI-E3H1) and chemical probe (PFI-7) were developed as ligands for the GID4 subunit of the human E3 ligase CTLH degradation complex, leveraging Pfizer's extensive compound library.

“We found that PFI-7 selectively engages GID4 in live cells, inhibiting Pro/N-degron binding, and is considered a suitable tool compound for use in cellular studies to investigate GID4 mediated recruitment to the CTLH complex.”, says Dr. Dominic D. G. Owens, SGC alumnus and co-author of the study.

Uncovering the GID4 interactome

The interactome of GID4 has never been determined using GID4 as a bait. The researchers used proximity-dependent biotinylation analysis to define the interactome of GID4, which complements the growing number of interactomes for human proteins.  “We were able to demonstrate a strong tendency for GID4 to interact broadly with distinct classes of nuclear proteins involved in RNA processing, chromatin, splicing and transcription.”, explains Dr. Maitland, Post Doctoral Fellow at SGC and co-author of the study.

PFI-7, however, facilitated the identification of proteins, or their interactors, that bind directly via the GID4 substrate binding pocket. The interactors lost upon treatment with PFI-7 were associated with gene ontology terms related to nucleic acid binding, ribosome biogenesis, and the nucleus, including RNA helicases DDX21 and DDX50. These interactors could represent candidate targets of the Pro/N-degron pathway and PFI-7 blocks these interactions. 

Additionally, PFI-7 treatment identified a distinct subset of proteins regulated by GID4, such as HMGCS1, a metabolic enzyme. Interestingly, several of the GID4 interacting proteins identified by PFI-7 are not substantially regulated at the protein level, while others are dependent on GID4 for protein-level regulation, possibly mediated through indirect interactions with the CTLH complex.

 “It is clear that there is much still to learn about the fundamental biology of the mammalian CTLH complex.” Dr. Arrowsmith explains, “PFI-7 will be invaluable for future investigations into the CTLH complex and in facilitating the development of targeted protein degradation strategies.”

Conclusion

The discovery of PFI-7 and its use in understanding the CTLH complex represents a significant advancement in the study of protein degradation. From an uncharacterized protein to a fully developed chemical probe, this journey exemplifies the mission of the SGC and Target 2035 to decode the dark proteome and identify pharmacological modulators for the entire human proteome. By leveraging the activity of the CTLH complex, researchers can design strategies to target and degrade disease-causing proteins, particularly in conditions where abnormal protein accumulation is a major factor, such as cancer and neurodegenerative disorders. As research continues, the collaboration between academia and industry will undoubtedly lead to further exploration of GID4 and the CTLH complex as an E3 ligase in targeted protein degradation. 

For more details, you can read the full publication here.

More information about PFI-7 can be found: https://pubmed.ncbi.nlm.nih.gov/38516600/