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What is a degron?
At the most fundamental level you can think of a degron as a sequence of amino acids or a structural motif that can facilitate the degradation of protein substrates. Researchers are interested in degrons because they provide insight into the way targeted protein degradation takes place naturally in human or other cells or how it can be engineered by new technologies like degron tags. Progress in the drug discovery and development of PROTACs (proteolysis-targeting chimeras), molecular glues or other emerging degrader technologies has broadened our understanding of degrons. These advances inform the discovery of new mechanisms that may deliver novel technologies and next generation therapeutics.
In this blog, we look at what degrons are, explore the ways that degrons can be used for targeted protein degradation applications, and give examples of how microplate readers can support research into natural and engineered degrons.
The emergence of degrons
Degrons are short amino acid sequences or structural motifs of proteins that signal the degradation of these structural or functional molecules by processes in the cell that lead to targeted protein degradation (Fig. 1). These amino acid sequences are often found in areas of proteins that are exposed at the protein surface which serve as a point of interaction with an E3 ligase or other start signal for the degradation of the protein. Abnormal proteins, which are misfolded or defective due to genetic mutations or faulty processes, are specifically targeted and eliminated by degradation systems involving ubiquitin ligases to maintain cellular homeostasis.
Distinguishing features of degrons
Primary degrons directly initiate degradation. Secondary degrons (or cryptic degrons) are latent protein motifs that only become active under certain conditions. Tertiary degrons refer to a three-dimensional structural feature or motif within a protein that may become active after protein misfolding, conformational changes, or the impact of binding a ligand. Tertiary degrons may initially be buried within the protein structure but become more available for binding after structural changes.
The first degrons to be discovered were located at the extreme N-terminus of proteins, a finding which initiated the study of the N-degron (formerly N-end rule) pathways, but only in the last few years has it emerged that a diverse set of C-degron pathways target similar degron motifs located at the extreme C-terminus of proteins.2
Collectively, a broad range of E3 ubiquitin ligases are involved in the recognition of a diverse array of terminal degron motifs within the ubiquitin proteasome system. These types of interactions can bring different degradative pathways into play and may influence a wide variety of cellular functions.
Researchers want to have a comprehensive understanding of how the recognition of specific degrons by E3 ubiquitin ligases drives selective protein degradation. Although our knowledge of degron motifs remains in its infancy, a wide array of E3 ligases are known to mediate degradation of their substrates through the selective recognition of these terminal degrons but in many cases the physiological roles of these pathways remain to be explored. What is clear is that they act on a diverse array of substrates and will impact a wide spectrum of cellular processes.
Genetic approaches that allow for the high-throughput identification of degron motifs and their cognate E3 ligase hold considerable promise in expanding our understanding of the degronome (the cellular degron landscape). The use of high-throughput methods to look at protein-protein interactions for degrons and their E3 ligases will also be crucial to reveal the significance of these interactions.
Tertiary degrons refer to a three-dimensional structural feature or motif within a protein that may become active after protein misfolding, conformational changes, or the impact of binding a ligand. Efficient degradation of proteins often depends on the type of degron present, as different degrons can influence the rate and way proteins are targeted and broken down within the cell.
Understanding these distinctions is crucial for developing targeted therapies and for advancing our knowledge of cellular processes.
The ubiquitin proteasome system and ubiquitin dependency of degrons
Degrons can be classified as either ubiquitin-dependent or ubiquitin-independent based on their interactions with the ubiquitin proteasome pathway.
In many cases, the exact mechanism by which a degron contributes to polyubiquitination is unknown. In such instances, degrons are considered ubiquitin dependent if their removal from a protein leads to less ubiquitination or conversely if their addition leads to more ubiquitination.
Naturally occurring degrons
Another important distinction for degrons is whether they occur naturally or are engineered into proteins. As mentioned earlier, some of the first degrons discovered were those in the N-terminus region of proteins (N-degrons).2 Early discoveries also revealed that proteins with sequences of arginine, lysine, phenylalanine, leucine and tryptophan were rapidly degraded in the cell. Sequences with methionine and glycine were more stable. Human proteins, particularly those with specific amino acid compositions at their C-termini, are also subject to degradation through C-degron pathways, with the scarcity of glycine and diGly motifs influencing their stability.
In time, other sequences at other locations within proteins were uncovered. Examples include proteins with PEST sequences (proline, glutamate, serine and threonine) and phospho-degrons. Membrane proteins, depending on their C-terminal topology, show varying susceptibility to the C-degron pathway, with differences in stability and degradation rates between those with inward-facing versus outward-facing C-termini. The list continues to grow as indicated by the development of resources like degronopedia, a web server for proteome-wide inspection of degrons that includes access to most of the reported degron sequences.Roles for degrons are emerging in cell cycle regulation, transcription, responses to stress and many other essential processes of the cell.
Degrons and protein quality control
Protein quality control is essential to maintain the homeostasis of the proteome and cell viability. In the context of homeostasis, naturally occurring degrons can serve as built-in quality control signals in proteins by allowing the cell to distinguish between functional and misfolded proteins. This ensures that protein homeostasis is maintained and helps to prevent the onset of misfunction and disease. Analyzing global protein stability is crucial in this context, as it helps to understand how different peptide sequences, such as the diGly motif, impact resistance to degradation pathways.
The quality control of targeted protein degradation in the cell at the level of degrons has consequences for the assembly and function of different protein complexes. In the study Recognition of the CCT5 di‐Glu degron by CRL4DCAF12 is dependent on TRiC assembly a team of researchers were able to discover a previously unknown role for the CRL4DCAF12 E3 ubiquitin ligase in overseeing the assembly of a TRiC chaperonin.3 TRiC has been linked to human pathologies such as cancers and neurodegenerative disease.
The researchers investigated whether C‐terminal degrons recognized by DCAF12 could trigger protein degradation and serve as signals for complex assembly. CCT5, a key subunit of the TRiC chaperonin, carries a di‐Glu degron at its C‐terminus. This degron becomes hidden once CCT5 assembles into TRiC. The CRL4DCAF12 ubiquitin ligase detects this degron when it is exposed to survey TRiC assembly and ensure that it functions correctly.
As part of the research, time-resolved fluorescence resonance energy transfer (TR-FRET) assays were set up to quantify the binding affinity of a CCT5 C-terminal peptide to DDB1-DCAF12. Spatial proximity resulted in fluorescence energy transfer giving a read out for binding. Competition assays were performed on BMG LABTECH’s PHERAstar® FSX microplate reader and the TR-FRET signal was plotted to calculate the half-maximal inhibitory concentration (IC50) assuming a single binding site.
In the study Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10 researchers looked at the recognition of a simple degron sequence for protein quality control and the evolution of Ala-tail proteolytic signaling.4
In this study, researchers investigated the role of C-end degrons, one of the more recently discovered classes of degrons, whose sequence diversity and interactions with E3 ligases are beginning to be elucidated.2 C-end degrons are naturally present in some proteins, may be exposed by proteolytic cleavage, can be generated by damage, or arise due to other modifications, as in processes like ribosome-associated quality control.
The paper looked at the relationship between C-end degrons and Ala-tail proteolytic signaling. The results provide further evidence to support the widely conserved role of Ala tails as a proteolysis tag and serve as a guide for future analyses across diverse organisms for protein modification and sensing mechanisms. As part of the study, an AlphaScreen proximity assay was used to look at the binding of different parts of the protein machinery (Pirh2 or KLHDC10) to the Ala-peptide. Competition assays were used to determine IC50 values. The AlphaScreen signal was measured at room temperature using BMG LABTECH’s PHERAstar FSX.
Opportunities for engineering targeted protein degradation with degrons
In addition to studying naturally occurring degrons, scientists are developing ways to engineer the presence of degrons in different proteins. By introducing new degrons into proteins researchers can control when and how proteins are degraded, which is useful to control protein stability, regulate different pathways in cells, and develop new therapeutic strategies. For instance, using stable proteins to cap putative C-degrons can alter their end-position dependency and effectively inactivate their function, highlighting the importance of the residues used for capping in experimental designs.
The topic of new approaches to drug discovery and development is further explored in “Developing next-generation therapeutics with targeted protein degradation”, an interview with Helen Harrison, Director of Screening at Amphista Therapeutics. The interview looks at emerging trends in degrader research, how targets for targeted protein degradation differ from conventional drug targets, and how microplate readers can support measurements of functional effects of protein degradation.
In the paper Continuous evolution of compact protein degradation tags regulated by selective molecular glues researchers developed a compact degron tag that was capable of engineering molecular glue interactions using diverse small molecules. Many of the tags used today that work with molecular glues are too large to label the genes that encode a cell’s native proteins or act too widely beyond the targeted protein. To overcome this limitation, researchers at the Broad Institute of MIT and Harvard University developed a continuous evolution platform called PACE that generates smaller degrons that form molecular glue complexes with the specificity needed to trigger depletion of the target protein. The compact degron is introduced into the cell’s genome by prime editing where it recruits the ubiquitin ligase cereblon. The PACE platform can be used to evolve molecular glue complexes with novel degrons.5
Molecular glue discovery for pre-defined targets is a major challenge in contemporary drug discovery. The field is limited by a lack of approaches that can exploit charged ligand-binding pockets, thus excluding a major fraction of ubiquitin ligases (E3s) that evolved to recognize exceedingly common acidic and basic degrons.
In the study Charged Molecular Glue Discovery Enabled by Targeted Degron Display a group of researchers describe a strategy to discover negatively charged molecular glues or c-Glues that induce proximity to ubiquitin ligases with similarly desirable properties.6
Using what they call a chemocentric molecular glue discovery strategy, the researchers discovered ZZ1, a BET-family (Bromodomain and Extraterminal Domain) protein degrader and a prodrug of a negatively charged glue (c-Glue). The study demonstrates a previously unrecognized capacity of YPEL5 to recruit GID/CTLH (glucose-induced degradation deficient/C-terminal to LisH complex) substrates. YPEL5 is a cereblon structural homolog and an essential non-Cullin ubiquitin ligase cofactor expressed in cancers of the bone marrow. Ternary complex formation for targeted protein degradation was determined by fluorescence polarization measurements on a CLARIOstar Plus microplate reader from BMG LABTECH.
GID is a substrate receptor involved in the recognition of Pro/N-degrons in the CTLH complex. CTLH is a multi-subunit ubiquitin ligase complex of interest since it is implicated in humans in a wide variety of cellular processes including autophagy, development, cell cycle regulation, and primary cilium function. Changes in CTLH complexes are also common in some cancers which makes them of interest for clinical studies.
New tools for degron research
Researchers are also exploiting the interactions of degrons with other molecules to develop new tools to study targeted protein degradation. One such tool employs a reporter system that uses two distinct fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein (RFP), to evaluate protein stability based on the degradation of GFP versus RFP.
In the study A chemical probe to modulate human GID4 Pro/N-degron interactions researchers developed PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4.7 Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.
The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to the CTLH complex as a potential mediator of targeted protein degradation. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences.
Degrons in action
Identifying and classifying a degron marks only the beginning of understanding the degradation process for its protein. Efficient degradation can be influenced by the competition for degradation resources, affecting gene expression and the degradation rates of co-expressed markers. What happens once a degron engages with an E3 ligase? As we saw earlier, one route for degradation is the ubiquitin proteasome system (Fig. 4). However, this is not the only option available and many emerging technologies are being developed for other pathways in the cell including the lysosomal pathway (see the BMG LABTECH blog Targeted protein degradation & next-generation degraders for further details). 
The degradation process of different proteins can be readily measured by cell-based assays on a microplate reader. In many cases, methods like HiBiT technology can be used to generate a luminescence readout (Fig. 5). Here HiBiT, a small 11-amino-acid peptide, is used to endogenously tag a target protein of interest using for example gene editing techniques like CRISPR-Cas9. Luminescence occurs when HiBiT interacts with its LgBiT counterpart. The treatment of cells with PROTAC leads to polyubiquitination and subsequent degradation of the protein of interest and the luminescent tag. The luminescent signal decreases as degradation proceeds (Fig. 5).
How do microplate readers contribute to degron research
Microplate readers are versatile tools for research into degrons and can be used to assess the efficacy of degron-mediated protein degradation. Moreover, screening for molecules that interact with degrons typically requires high-throughput capabilities. Here microplate readers help increase throughput and decrease the need for manual intervention.
Over the years a range of technologies have been used to study the different steps of the ubiquitin-proteasome pathway and ubiquitin-dependent protein degradation (Fig. 6). These have been in many cases time-consuming, expensive and had a large footprint (Fig. 6; grey). The use of cell-based assays and microplate readers now permits all the steps in the ubiquitin-proteasome pathway to be measured much more efficiently and at lower cost and demand on resources (Fig. 6; purple). This approach can also be extended to the lysosomal pathway and many of the emerging technologies for targeted protein degradation.
An optimized live cell workflow on a microplate reader is capable of screening all the steps of the ubiquitin-proteasome targeted protein degradation pathway (for further details see the eBook Next-generation therapeutics: PROTACs and molecular glues).
Luminescence and other luminescence-derived technologies are powerful ways to look at protein-ligand binding for degron-related research. NanoBRET® and HiBiT enable binding and ubiquitination to be measured in live cells. This is further empowered by microplate readers equipped with temperature incubation and atmospheric control. Instruments like the CLARIOstar® Plus even allow long-term kinetic experiments to be run in the plate reader while keeping a physiological environment for cells.
Fluorescence polarization assays can also be used to study the binding activity of degrons to their targets and the kinetics of interactions (Fig. 7). Changes in fluorescence polarization due to fluorescently labeled drug candidates binding to target proteins may be measured. This can provide information about the binding strength and kinetics of the interaction between the target protein and degrader.
The application note Ubiquitination monitoring in real-time: the fluorescence polarization-based method UbiReal describes a fluorescence polarization method that can be used to track all stages of ubiquitin conjugation and deconjugation in real time. The approach is suitable for high throughput formats and fluorescence polarization is used to measure fluorescently labeled ubiquitin. All stages of the ubiquitination cycle can be monitored using UbiReal (Fig. 8).
In addition, high-throughput screening techniques can be used to prioritize potential degraders and microplate readers are a useful tool that scales to the needs of the screen.
What is the preferred BMG LABTECH microplate reader for specific needs and applications related to degron research? BMG LABTECH offers a range of detection devices for sensitive absorbance, fluorescence and luminescence measurements.
The PHERAstar FSX was specifically conceived for screening campaigns and is your go-to reader for high-performance high-throughput investigations in molecular glue research.
Both the VANTAstar® and CLARIOstar Plus allow for wavelength scanning and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter MonochromatorsTM and different filter options. In addition, they are recommended for live cell-based assays as they can be equipped with the Atmospheric Control Unit for CO2 and O2 regulation in the instrument.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus and PHERAstar FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection.
Collectively, these multi-mode readers combine high performance with miniaturized assays, short measurement times, and offer considerable savings on materials and other resources.
For more information on targeted protein degrader experiments and more ways to evaluate molecular glues, PROTACs and other degraders check out this scientific talk:
Configure your microplate reader and get an initial recommendation!
References
- Harris TJ Jr, Trader DJ. Exploration of degrons and their ability to mediate targeted protein degradation. RSC Med Chem. 2025 Jan 1. doi: 10.1039/d4md00787e.
- Timms RT, Koren I. Tying up loose ends: the N-degron and C-degron pathways of protein degradation. Biochem Soc Trans. 2020 Aug 28;48(4):1557-1567. doi: 10.1042/BST20191094.
- Pla-Prats C, Cavadini S, Kempf G, Thomä NH. Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly. EMBO J. 2023 Feb 15;42(4):e112253. doi: 10.15252/embj.2022112253. Epub 2023 Jan 30.
- Patil PR, Burroughs AM, Misra M, Cerullo F, Costas-Insua C, Hung HC, Dikic I, Aravind L, Joazeiro CAP. Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10. Cell Rep. 2023 Sep 26;42(9):113100. doi: 10.1016/j.celrep.2023.113100.
- Mercer JAM, DeCarlo SJ, Roy Burman SS, Sreekanth V, Nelson AT, Hunkeler M, Chen PJ, Donovan KA, Kokkonda P, Tiwari PK, Shoba VM, Deb A, Choudhary A, Fischer ES, Liu DR. Continuous evolution of compact protein degradation tags regulated by selective molecular glues. Science. 2024 Mar 15;383(6688):eadk4422. doi: 10.1126/science.adk4422.
- Zhe Zhuang, Woong Sub Byun, Jakub Chrustowicz et al. Charged Molecular Glue Discovery Enabled by Targeted Degron Display bioRxiv 2024.09.24.614843; doi: https://doi.org/10.1101/2024.09.24.614843
- Owens DDG, Maitland MER, Khalili Yazdi A, Song X, Reber V, Schwalm MP, Machado RAC, Bauer N, Wang X, Szewczyk MM, Dong C, Dong A, Loppnau P, Calabrese MF, Dowling MS, Lee J, Montgomery JI, O'Connell TN, Subramanyam C, Wang F, Adamson EC, Schapira M, Gstaiger M, Knapp S, Vedadi M, Min J, Lajoie GA, Barsyte-Lovejoy D, Owen DR, Schild-Poulter C, Arrowsmith CH. A chemical probe to modulate human GID4 Pro/N-degron interactions. Nat Chem Biol. 2024 Sep;20(9):1164-1175. doi: 10.1038/s41589-024-01618-0.
