Phage Display for Novel E3 Ligase and Deubiquitinase Substrates
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The ubiquitin-proteasome system (UPS) is the primary mechanism for the controlled degradation of proteins in eukaryotic cells. At the heart of this system are two opposing forces: the E3 ligase enzymes that tag proteins for destruction, and the deubiquitinase (DUB) enzymes that remove these tags to stabilize proteins. Balancing this equation is critical for cell survival. Identifying the specific substrates of these enzymes is considered the ultimate goal in modern drug discovery, especially for developing targeted protein degradation (TPD) therapeutics. However, the transient nature of these enzyme-substrate interactions makes them notoriously difficult to capture. Creative Biolabs' phage display for novel E3 ligase and deubiquitinase substrates service offers a robust, high-throughput solution for mapping these elusive interactions. We utilize advanced phage display technology to screen your target enzyme against comprehensive human cDNA or peptide libraries. This approach stabilizes weak interactions in vitro, enabling us to identify novel substrates that traditional methods, such as mass spectrometry, often overlook due to their low abundance or rapid turnover. We offer a streamlined yet comprehensive discovery process designed to characterize the set of degradation-related features of your target E3.:
- We perform unbiased screening of E3 ligases and DUBs against genome-wide cDNA libraries to identify physiological binding partners.
- Our platform maps specific recognition motifs using high-diversity peptide libraries.
- We facilitate E3 ligase substrate discovery to identify new handles for PROTAC development.
- The service enables DUB substrate identification to understand protein stabilization mechanisms.
- We deliver a ranked list of high-confidence candidates validated by bioinformatics.
- Our team supports downstream validation to confirm biological relevance.
This specialized substrate discovery platform is a key application of our broader Phage Display Protein Interaction Mapping Service, leveraging our core expertise in molecular recognition to decode the complex language of cellular signaling and stability.
Challenges in Ubiquitin Ligase and DUB Research
To understand disease and develop new drugs, it is essential to comprehend how protein levels are regulated. The ubiquitination pathway is the central regulator of this process. It involves a cascade of enzymes (E1, E2, and E3) that covalently attach ubiquitin molecules to a substrate. The E3 ligase provides specificity, serving as the matchmaker that recognizes the target protein. Conversely, a DUB acts as an editor, cleaving the ubiquitin chain to rescue the protein from the proteasome or to alter its signaling function. Dysregulation of this machinery leads to cancer, neurodegeneration, and immune disorders. Consequently, these enzymes are prime targets for drug discovery. Specifically, the field of targeted protein degradation, including PROTAC target development, relies entirely on knowing which E3 ligase pairs with which substrate. Despite their importance, identifying the substrates of a specific E3 or DUB is exceptionally challenging due to inherent biological hurdles:
- Transient Interactions: The interaction between an E3 ligase and its substrate is often a "hit-and-run" event. Once ubiquitination occurs, the complex dissociates rapidly, and the substrate is destroyed. This transience makes capture by co-immunoprecipitation (Co-IP) difficult.
- Low Abundance: Many regulatory substrates exist at very low physiological levels. Mass spectrometry-based proteomics is biased toward abundant proteins, often failing to detect the rare but critical regulators.
- Weak Affinity: The binding affinity between an E3 and its recognition motif is often in the micromolar range, which is biologically sufficient for catalysis but structurally unstable for isolation.
- Promiscuity vs. Specificity: A single E3 may have dozens of substrates, and multiple DUBs may regulate a single substrate. Untangling this web requires a method that can screen the entire proteome in a controlled environment.
Fig.1 Mechanism of protein substrate ubiquitination.1
Phage display overcomes these challenges. By displaying billions of potential substrate fragments on phages and incubating them with the enzyme in vitro (often supplemented with essential reaction components, such as E1, E2, and ubiquitin, to stabilize relevant complexes), we can capture weak and transient binders. It is important to note that while the phage display platform identifies high-affinity binders, confirming them as true ubiquitination substrates requires validation in a complete enzymatic system.
Specialized Substrate Discovery Services for E3s and DUBs
We have tailored our phage display platform to address the specific biochemical requirements of ubiquitin-related enzymes, providing clear answers regarding substrate specificity and recognition motifs.
E3 Ligase Substrate Profiling
This service is our flagship solution for PROTAC target discovery and broad substrate mapping. We screen your specific E3 ligase (such as VHL, CRBN, MDM2, or novel orphan E3s) against a high-complexity phage display cDNA Library to identify the full repertoire of proteins that the E3 binds to and may potentially degrade. By determining the candidate physiological binding partners, we provide a list of putative substrates that can be exploited for targeted protein degradation strategies or used to understand the biological function of an orphan E3 ligase.
DUB Substrate Identification
Inhibiting a DUB can be a powerful therapeutic strategy to destabilize specific oncoproteins, but this requires knowing exactly which proteins the DUB protects. Our service addresses this by screening the DUB against cDNA libraries to determine its specific interactome. To capture these transient interactions, we frequently utilize catalytically inactive "substrate-trap" mutants of the DUB, which bind to the substrate but cannot cleave the ubiquitin chain, thereby creating a stable complex that is ideal for phage capture and subsequent identification of the DUB substrate. Validation with wild-type DUBs in cellular assays is recommended to confirm physiological relevance.
Degron Motif Discovery
Many E3 ligases recognize a short, linear amino acid sequence on the substrate, known as a degron. To define this precise consensus sequence, we utilize random peptide libraries (linear or constrained) to screen against the ligase. Identifying the specific degron motif provides a powerful predictive tool, allowing you to search the entire proteome bioinformatically for novel substrates that contain this sequence, thereby expanding your understanding of the ligase's regulatory reach.
Screening for Molecular Glues
Some E3 ligases require the presence of a small molecule or molecular glue to alter their surface and recruit substrates that they would not naturally bind. To support this cutting-edge area of drug discovery, we perform phage panning in the direct presence of your candidate molecular glue. This specialized screening format allows us to identify "neo-substrates" that are recruited only when the compound is present, providing critical data for the development of novel therapeutics that hijack the ubiquitination pathway.
Phage Display Substrate Screening Workflow
We employ a rigorous, five-stage process designed to filter out noise and deliver high-confidence, biologically relevant candidates.
The project initiates with a strategic consultation with our Ph.D. scientists to strictly define your goals, whether it is E3 ligase substrate mapping or DUB profiling. A critical component of this phase is the target design and preparation; for E3s, we often select specific substrate-binding domains or full-length proteins stabilized with E2-ubiquitin conjugates to preserve native conformation. For DUBs, we design and produce "substrate-trap" mutants to ensure the stability of protein complexes. We determine the optimal expression system and immobilization strategy to maintain the native conformation of your target enzyme throughout the screening process.
Based on your scientific question, we select the optimal library strategy, utilizing our Phage Display cDNA Library for mapping potential physiological interactors or peptide libraries for degron definition. We then execute 3-5 rounds of our Library Screening & Biopanning protocols, where the library is incubated with your immobilized target enzyme. Appropriate negative controls and competitive elution strategies are employed to reduce false positives and enrich for sequences that specifically recognize the E3 or DUB.
Rather than relying on the limited scope of picking a few clones, we employ Next-Generation Sequencing (NGS) to analyze the entire pool of enriched phages. This high-throughput approach allows us to generate millions of reads, providing a comprehensive and quantitative landscape of the binding population. This depth of coverage is essential for detecting low-abundance novel substrates and rare binding events that would be statistically invisible to lower-resolution sequencing methods.
Our bioinformatics team processes the massive NGS dataset to transform raw reads into actionable biological insights. This involves rigorous data cleaning and statistical filtering to remove background noise, followed by mapping the DNA sequences back to the human proteome to identify the gene IDs of potential substrates. For peptide screens, we utilize advanced clustering algorithms to identify conserved amino acid consensus motifs required for ubiquitination or deubiquitination recognition, thereby distinguishing accurate signals from random binding.
The final deliverable is a comprehensive report that contains a ranked list of hits, domain mapping, and motif analysis, directly answering your research question. We transparently discuss potential limitations, such as the need for post-translational modifications that may be absent in phage display. For clients seeking further confirmation, we offer optional downstream validation services. These services involve testing the top E3 ligase substrate or DUB targets using biochemical assays, such as in vitro ubiquitination or Co-IP, to confirm that the identified binding leads to functional regulation in a biological context.
Discuss Your Project
Core Technologies Driving Substrate Discovery
We utilize a suite of advanced platforms to ensure the success of your substrate discovery project.
Advanced Screening Platforms
To ensure we can successfully screen any target, regardless of its structural complexity, we operate two distinct screening platforms. Our in vitro protein-based phage display screening platform is the industry standard for purified E3s and DUBs, utilizing specific tags for clean immobilization and optimized buffers containing the necessary co-factors. For more complex targets, such as multi-subunit E3 ligase complexes that require a cellular environment for proper folding, our In Vitro Cell-Based Phage Display Screening Platform enables us to capture biologically relevant interactions in their native state.
Comprehensive Phage Display Libraries
Our extensive library portfolio allows us to tailor the discovery scope precisely to your needs. We utilize Phage Display cDNA Libraries constructed from relevant tissues to ensure identified potential substrates are biologically expressed in your system. Complementing this, our Phage Display Peptide Libraries are essential for mapping minimal degron motifs, while our Antibody and Scaffold libraries provide tools to block or stabilize these interactions. We can even generate Custom Libraries from specific source species to match your model organism.
Enabling Technologies
To distinguish an accurate signal from experimental noise, we leverage our Phage Display NGS Service. This technology provides the deep resolution required to identify rare substrates among billions of non-binders, ensuring that the candidates we report are backed by robust statistical evidence.
Advantages of Our Phage Display Platform
Comprehensive Library Options
We offer cDNA, Peptide, Antibody, and Scaffold libraries, allowing us to precisely match the tool to your specific E3 ligase substrate discovery needs.
Advanced Screening Platforms
We offer both protein-based and cell-based screening, ensuring we can handle a diverse range of enzyme classes.
Expert Scientific Support
You work with a dedicated team of Ph.D. scientists with deep expertise in the ubiquitination pathway. We guide you from target design to data interpretation.
High Throughput & Speed
We screen billions of candidates simultaneously. Combined with NGS, this accelerates the PROTAC target discovery timeline significantly compared to traditional proteomics.
Applications in TPD and Drug Discovery
The identification of novel substrates opens immediate doors for drug development and basic research.
Accelerating PROTAC and Molecular Glue Development
The field of targeted protein degradation is revolutionizing the field of medicine. To design a PROTAC, you need to recruit an E3 ligase to a target protein. However, we currently utilize only a tiny fraction (e.g., CRBN, VHL) of the ~600 human E3 ligases. By using our service to map the natural substrates of orphan E3 ligases, we can identify new E3 ligases that are suitable for PROTAC recruitment, thereby expanding the TPD toolbox.
Validating DUB Inhibitors
Inhibiting a DUB is a strategy to force the degradation of a pathogenic protein. However, to know if a DUB is a good target, you must understand what it stabilizes. Our DUB substrate identification service helps you predict the therapeutic effect of DUB inhibition.
Understanding Disease Mechanisms
Many oncogenes and tumor suppressors are regulated by protein degradation. Mapping the E3s and DUBs that control their levels provides critical insight into cancer progression and reveals new intervention points in the ubiquitination pathway.
The interactions defining the ubiquitination landscape are complex, transient, and vital. Don't let the limitations of traditional screening hold back your research. Our phage display service offers the sensitivity and depth required to identify novel E3 ligase and deubiquitinase substrates with confidence. Contact us today to discuss your ubiquitin-related target and receive a customized project proposal tailored to your needs.
FAQs
Can you work with any type of E3 ligase (RING, HECT, RBR)?
Yes. We have experience working with all major classes of E3 ligases. The key is proper target preparation. For RING ligases, which often act as scaffolds for E2 enzymes, we recommend including the complete E1-E2-Ubiquitin-ATP system in the screening buffer to fully reconstitute the native ubiquitination environment. For HECT and RBR ligases, which form a covalent intermediate with ubiquitin, we prioritize screening with full-length proteins or constructs containing the catalytic domain (often with catalytically inactive mutations to prevent auto-ubiquitination) to preserve critical substrate recognition conformations that rely on the catalytic cysteine.
How do you handle the specificity of Deubiquitinases (DUBs)?
DUBs can be promiscuous, often recognizing the ubiquitin chain rather than the protein to which it is attached, but many also exhibit specific recognition for the substrate protein itself. To find substrates that interact directly with the DUB, we usually use "substrate-trap" mutants (e.g., Cys→Ser/Ala). These are DUB variants with a point mutation in the active site that enhances interaction stability. They can bind the ubiquitinated substrate but cannot cleave the chain, effectively "locking" the substrate to the DUB. To ensure biological relevance and rule out non-physiological interactions caused by the mutation, we validate these hits using wild-type DUBs in functional assays, such as co-immunoprecipitation (Co-IP) or deubiquitination experiments.
Is this service useful for PROTAC and TPD research?
Absolutely. This is one of the primary applications. To develop a PROTAC, you need to know which E3 ligase can be recruited to degrade your target of interest, or conversely, which E3s are available in a specific tissue. By profiling the substrate specificity of various E3s and combining this with expression and localization data, we help you identify potential new PROTAC target pairs and novel E3 ligases that can be exploited for targeted protein degradation. The identified candidates are then subjected to rigorous cellular degradation assays to confirm their suitability for PROTAC development.
Reference:
- Kubaichuk, Kateryna, and Thomas Kietzmann. "Involvement of E3 Ligases and Deubiquitinases in the Control of HIF-α Subunit Abundance." Cells 8.6 (2019): 598. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.3390/cells8060598
Resources
Please kindly note that our services can only be used to support research purposes (Not for clinical use).
