Recombinant Chicken E3 UFM1-protein ligase 1 (UFL1), partial

What is UFL1 and what is its role in the UFM1 conjugation pathway?

UFL1 (UFM1-protein ligase 1) is a novel type of E3 ligase specific to the UFM1 conjugation system. Unlike other E3 ligases in ubiquitin and ubiquitin-like modifier systems, UFL1 has no obvious sequence homology to any other known E3s . UFL1 functions as the final enzyme in the three-step enzymatic cascade that facilitates the covalent attachment of UFM1 to target proteins, a process known as ufmylation. The pathway begins with UFM1 activation by the E1-activating enzyme UBA5, followed by transfer to the E2-conjugating enzyme UFC1, and finally, UFL1 mediates the transfer of UFM1 to specific substrates .

The UFM1 conjugation system is conserved in multicellular organisms and is implicated in various cellular processes including endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER (ER-RQC), and ER-phagy . Additionally, recent research has identified a role for UFL1 in ribosomal DNA double-strand break repair, suggesting its importance in maintaining genomic stability .

How does UFL1 differ structurally and functionally from other E3 ligases?

UFL1 represents a unique class of E3 ligases with no apparent sequence homology to traditional E3 ligases found in the ubiquitin system or other ubiquitin-like modifier systems . While conventional E3 ligases typically contain domains such as RING finger, HECT, or U-box domains, UFL1's structure appears distinct.

Functionally, UFL1 forms a complex with UFBP1 (UFM1-binding protein 1), which together constitute the functional E3 ligase complex for UFM1 . This complex interacts with UFC1 (the E2 enzyme) and subsequently with CDK5RAP3, which serves as an adaptor for the ufmylation of specific substrates such as the ribosomal subunit RPL26 . The unique structural arrangement allows UFL1 to facilitate the transfer of UFM1 from UFC1 to target proteins, accelerating the formation of isopeptide bonds between UFM1's C-terminal glycine and lysine residues on substrate proteins.

What are the known cellular substrates of UFL1-mediated ufmylation?

Several cellular substrates for UFL1-mediated ufmylation have been identified through biochemical and proteomic approaches. One of the first discovered targets was C20orf116 (also known as UFBP1), which forms a covalent conjugate with UFM1 . This conjugation is reversible and can be cleaved by UFM1-specific proteases, demonstrating the dynamic nature of ufmylation .

More recent research has identified the ribosomal protein RPL26 as a substrate for ufmylation . The ufmylation of RPL26 on the 60S ribosomal subunit appears to play a critical role in ribosome-associated quality control at the endoplasmic reticulum. Upon disome formation (which occurs during translational stalling), the UFL1-UFBP1 E3 complex associates with ufmylated RPL26 through the UFM1-interacting region of UFBP1 .

Other potential targets have been identified through mass spectrometry-based approaches, though further validation is required to confirm their physiological relevance.

What expression systems are most effective for producing recombinant chicken UFL1?

When producing recombinant chicken UFL1, researchers should consider several expression systems based on the intended application. For structural studies and in vitro biochemical assays, bacterial expression systems using E. coli can provide high yields of protein. Based on the literature, MBP (maltose-binding protein) fusion tags have been successfully used to express UFL1 and its mutants in E. coli . The MBP tag enhances solubility and facilitates purification using amylose resin affinity chromatography.

For studies requiring post-translational modifications or proper folding of complex domains, eukaryotic expression systems are preferable. Mammalian expression systems (such as HEK293 cells) have been successfully used to express tagged versions of UFL1. For instance, 3× FLAG and GFP tags introduced at the N-terminus of UFL1 and expressed using the pIRESpuro3 vector system have yielded functional protein .

When designing expression constructs, consider the following approach:

• For bacterial expression: Clone the chicken UFL1 coding sequence into pMAL vectors for MBP fusion.

• For mammalian expression: Subclone UFL1 into pIRESpuro3 with appropriate tags (FLAG, GFP) for detection and purification.

• Include appropriate protease cleavage sites if tag removal is desired.

What purification strategies yield the highest purity and activity for recombinant UFL1?

Purification of recombinant UFL1 requires a strategic approach to maintain both purity and enzymatic activity. Based on published methodologies, the following multi-step purification procedure is recommended:

For MBP-tagged UFL1 from bacterial systems:

• Cell lysis in TN buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl) with protease inhibitors.

• Affinity chromatography using amylose resin, which binds the MBP tag.

• Elution with 10 mM maltose in elution buffer .

• Optional tag removal using specific proteases if the experimental design requires untagged protein.

• Size exclusion chromatography to remove aggregates and achieve higher purity.

For FLAG-tagged UFL1 from mammalian systems:

• Cell lysis in TNE buffer (10 mM Tris-Cl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA) with protease inhibitors.

• Immunoprecipitation with anti-FLAG antibody conjugated to agarose beads.

• Stringent washing to remove non-specific interactions.

• Elution using FLAG peptide competition .

For both systems, additional ion-exchange chromatography may help remove contaminants with different charge properties. Activity assessments should be performed at each purification step to ensure the final product retains enzymatic function.

How can I design deletion mutants of UFL1 to study domain functionality?

Designing deletion mutants of UFL1 is an effective strategy to dissect domain functionality. Based on previous research, the following approach can be employed:

• Conduct bioinformatic analysis of chicken UFL1 sequence to identify conserved domains and structural motifs.

• Design a series of deletion constructs that systematically remove different regions, following a strategy similar to the mutants described in the literature (Ufl1 M1 through M5) .

• Generate PCR primers with appropriate restriction sites for directional cloning.

• Express the deletion mutants with identical tags (e.g., MBP or FLAG) to ensure comparable detection and purification.

A recommended panel of UFL1 deletion mutants based on human UFL1 studies would include:

• N-terminal region (amino acids 1-212)

• Middle region (amino acids 213-794)

• C-terminal region (amino acids 453-794)

• N-terminal and middle regions combined (amino acids 1-452)

• Construct lacking only the C-terminal region (amino acids 1-654)

These constructs should be validated by sequencing and expression testing before functional characterization. Each mutant can then be assessed for its ability to interact with UFC1, bind substrates, and catalyze ufmylation to delineate the functional importance of each domain.

What in vitro assays can demonstrate the E3 ligase activity of recombinant UFL1?

Several in vitro assays can effectively demonstrate the E3 ligase activity of recombinant UFL1. These methodologies enable researchers to assess various aspects of UFL1 function, from protein-protein interactions to catalytic activity:

• Pull-down assay for E2 interaction:

  • Mix purified MBP-UFL1 (or mutants) with recombinant UFC1 in TN buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl) for 3 hours at 4°C.

  • Precipitate complexes using amylose resin.

  • Wash three times with ice-cold TNE buffer.

  • Elute bound proteins with 10 mM maltose.

  • Analyze by SDS-PAGE followed by immunoblotting with anti-UFC1 antibody and Coomassie staining .

• In vitro ufmylation assay:

  • Combine purified E1 (UBA5), E2 (UFC1), E3 (UFL1), mature UFM1, ATP, and substrate protein (e.g., C20orf116 or RPL26).

  • Incubate the reaction mixture at 37°C for 1-2 hours.

  • Analyze by SDS-PAGE and immunoblot using anti-UFM1 antibody to detect the formation of UFM1-substrate conjugates.

  • Include controls lacking individual components to confirm specificity.

• Thioester formation assay:

  • Similar to the ufmylation assay but analyzed under non-reducing conditions to preserve thioester bonds.

  • Compare reactions under reducing and non-reducing conditions to distinguish between thioester intermediates and isopeptide-linked final products.

These assays can be complemented with kinetic measurements to determine reaction rates and efficiency, providing quantitative assessment of UFL1 activity and the effects of mutations or inhibitors.

How can I study the interaction between UFL1 and its binding partners?

To characterize interactions between UFL1 and its binding partners (UFC1, UFBP1, CDK5RAP3, and substrates), several complementary approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of UFL1 and potential binding partners in mammalian cells.

  • Lyse cells in TNE buffer (10 mM Tris-Cl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA) with protease inhibitors.

  • For interactions involving UFM1-conjugated proteins, denature lysates by boiling in 1% SDS-containing TNE buffer, then dilute 10-fold with SDS-free TNE buffer before immunoprecipitation .

  • Immunoprecipitate using antibodies against the tag or the protein of interest.

  • Analyze precipitated complexes by immunoblotting for interacting partners.

• Pull-down assays with recombinant proteins:

  • Express and purify recombinant UFL1 and binding partners with different tags.

  • Mix proteins in appropriate buffer conditions and isolate complexes using affinity resins.

  • Analyze bound proteins by SDS-PAGE and immunoblotting or mass spectrometry.

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse UFL1 and potential binding partners to complementary fragments of a fluorescent protein.

  • Express constructs in mammalian cells and analyze fluorescence reconstitution, which indicates protein-protein interaction.

  • This method provides spatial information about where in the cell the interaction occurs.

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize purified UFL1 on a sensor chip or biosensor.

  • Flow solutions containing potential binding partners over the immobilized protein.

  • Measure binding kinetics (association and dissociation rates) and calculate affinity constants.

These methods provide complementary information about the qualitative and quantitative aspects of UFL1 interactions, helping to build a comprehensive understanding of UFL1 function within the ufmylation pathway.

What cellular assays can evaluate the physiological role of UFL1 in chicken cells?

To investigate the physiological functions of UFL1 in chicken cells, researchers can employ several cellular assays that probe different aspects of UFL1-mediated processes:

• CRISPR/Cas9-mediated knockout or knockdown studies:

  • Design guide RNAs targeting chicken UFL1.

  • Assess effects on cell viability, proliferation, and morphology.

  • Compare fitness between control and UFL1-deficient cells, particularly under stress conditions (as demonstrated in the competitive growth assay for human cells) .

• ER stress response assays:

  • Treat control and UFL1-depleted cells with ER stress inducers (tunicamycin, thapsigargin).

  • Measure expression of ER stress markers (BiP, CHOP, XBP1 splicing) by qRT-PCR and Western blotting.

  • Assess cell survival under ER stress conditions.

• Ribosome-associated quality control assessment:

  • Use reporter constructs containing ribosome-stalling sequences.

  • Compare degradation efficiency of these reporters between control and UFL1-deficient cells.

  • This approach can reveal UFL1's role in ER-RQC, similar to findings in mammalian systems .

• DNA damage response studies:

  • Induce DNA damage, particularly in ribosomal DNA.

  • Evaluate nucleolar segregation using markers like TCOF1.

  • Quantify rRNA transcription using EU incorporation or Northern blotting, as these processes are affected by UFL1 deficiency in human cells .

• Immunofluorescence localization:

  • Determine the subcellular localization of UFL1 and potential co-localization with ER markers, nucleoli, or DNA damage sites.

  • This can provide insights into the spatial regulation of UFL1 functions.

These cellular assays can reveal the conservation of UFL1 functions between chicken and mammalian systems, as well as potentially identify species-specific roles or regulation mechanisms.

How can I enhance the solubility of recombinant UFL1 during expression and purification?

Enhancing the solubility of recombinant UFL1 requires optimizing several parameters during expression and purification. The following strategies can help address common solubility issues:

• Expression tag selection:

  • The use of MBP (maltose-binding protein) as a fusion tag has proven effective for UFL1 expression in E. coli .

  • Alternative solubility-enhancing tags include SUMO, Thioredoxin, or GST, which can be tested if MBP fusion proves insufficient.

• Expression conditions optimization:

  • Lower induction temperature (16-18°C) can significantly improve protein folding and solubility.

  • Reduce inducer concentration (IPTG) to slow protein production and allow proper folding.

  • Extend expression time (overnight or longer) at lower temperatures.

  • Test different media formulations, including enriched media or those containing osmolytes like sorbitol or betaine.

• Co-expression strategies:

  • Co-express UFL1 with its binding partners (UFBP1, UFC1) to promote complex formation and stability.

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J/GrpE) to assist proper folding.

• Buffer optimization during purification:

  • Include glycerol (10-15%) to stabilize protein structure.

  • Test various salt concentrations (150-500 mM NaCl) to screen for optimal solubility.

  • Add mild detergents (0.01-0.05% Triton X-100) to prevent aggregation.

  • Include reducing agents (DTT or TCEP) to maintain cysteine residues in reduced state.

• Partial truncation approach:

  • Design constructs that remove potentially problematic regions while retaining functional domains, similar to the Ufl1 deletion mutants described in the literature .

Implementing these strategies systematically, preferably in a factorial design experiment, can help identify optimal conditions for producing soluble and active recombinant UFL1.

How can I troubleshoot issues with UFL1 enzymatic activity in in vitro assays?

When encountering problems with UFL1 enzymatic activity in in vitro assays, systematic troubleshooting can help identify and resolve issues:

• Protein quality assessment:

  • Verify protein integrity by SDS-PAGE and mass spectrometry.

  • Check for degradation or truncation products that might lack key domains.

  • Assess protein folding using circular dichroism or thermal shift assays.

• Reaction conditions optimization:

  • Test different buffer compositions (pH 6.5-8.5, various salt concentrations).

  • Optimize incubation temperature (25-37°C) and time (30 minutes to 2 hours).

  • Vary ATP concentrations (1-5 mM) and include magnesium (2-10 mM MgCl₂).

  • Consider adding reducing agents (DTT or TCEP) to maintain active site cysteines.

• E1 and E2 enzyme quality:

  • Ensure UBA5 (E1) and UFC1 (E2) are active by performing thioester formation assays.

  • Consider using freshly prepared enzymes as activity may decrease with storage.

  • Titrate E1 and E2 concentrations to find optimal ratios with UFL1.

• Complete reaction component verification:

  • Confirm all necessary components are present: E1, E2, E3 (UFL1), mature UFM1 (with exposed C-terminal glycine), ATP, and substrate.

  • For UFM1, ensure the C-terminal processing is complete to expose the critical glycine residue.

• E3 complex formation consideration:

  • UFL1 functions in complex with UFBP1, and the complete E3 ligase activity may require both proteins .

  • Consider including recombinant UFBP1 in reaction mixtures if it's absent.

• Detection method sensitivity:

  • Ensure antibody quality and specificity for detecting UFM1 conjugates.

  • Consider more sensitive detection methods like fluorescently labeled UFM1.

If activity remains problematic, analyzing the activity of UFL1 deletion mutants can help identify which domains are critical for function , potentially guiding the design of more stable or active constructs.

What are common pitfalls in generating UFL1 knockout or knockdown models in avian systems?

Generating UFL1 knockout or knockdown models in avian systems presents several challenges that researchers should anticipate and address:

• Potential lethality:

  • Complete UFL1 knockout may be lethal during development, as suggested by fitness defects observed in UFL1-deficient human cells .

  • Consider conditional knockout systems (e.g., Cre-loxP) or inducible shRNA approaches to circumvent developmental lethality.

• Guide RNA design for CRISPR/Cas9:

  • Chicken genome annotation may be less comprehensive than mammalian models, complicating effective guide RNA design.

  • Design multiple guide RNAs targeting different exons, preferably early coding regions.

  • Validate guide RNA efficiency using in vitro cleavage assays before cellular application.

• Delivery methods in avian cells:

  • Transfection efficiency can be lower in primary avian cells compared to established mammalian lines.

  • Optimize transfection protocols specifically for the avian cell type being used.

  • Consider viral delivery systems (lentivirus or adeno-associated virus) for improved efficiency.

• Verification of knockout/knockdown:

  • Develop reliable detection methods for chicken UFL1 (antibodies, qRT-PCR primers).

  • Verify knockout at both genomic (sequencing) and protein (Western blot) levels.

  • Screen multiple clones due to potential mosaicism in CRISPR-edited cells.

• Compensation by related pathways:

  • Monitor potential upregulation of other protein modification systems that might compensate for UFL1 loss.

  • Consider acute knockdown models to minimize compensatory adaptations.

• Off-target effects:

  • Validate phenotypes with rescue experiments using wild-type UFL1 expression.

  • Use multiple independent knockout/knockdown lines to confirm phenotypes.

• Cell culture versus in vivo models:

  • Phenotypes in cell culture may not fully recapitulate the complexity of in vivo functions.

  • When feasible, complement in vitro studies with in ovo approaches to assess developmental roles.

Addressing these challenges requires careful experimental design and validation at multiple levels to ensure the generation of reliable UFL1-deficient avian models.

How should I interpret changes in ufmylation patterns in UFL1 mutant studies?

Interpreting changes in ufmylation patterns in UFL1 mutant studies requires careful analysis and consideration of multiple factors:

This multi-faceted analysis approach will help distinguish between direct effects of UFL1 mutations on ufmylation versus secondary consequences, providing deeper insights into UFL1 function.

What statistical approaches are most appropriate for analyzing UFL1 functional data?

Choosing appropriate statistical approaches for analyzing UFL1 functional data depends on the experimental design and data characteristics. Here are recommended statistical methods for different types of UFL1-related experiments:

• For biochemical assays comparing wild-type and mutant UFL1 activity:

  • Two-sample t-tests (paired or unpaired) for comparing means between two groups.

  • ANOVA with post-hoc tests (Tukey's, Bonferroni) when comparing multiple mutants or conditions.

  • Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) if data doesn't meet normality assumptions.

  • Use technical and biological replicates (n≥3) to ensure robust statistical inference.

• For cell-based phenotypic assays:

  • In competitive growth assays comparing UFL1 knockout to control cells:

    • Log-ratio analysis of relative cell proportions over time.

    • Linear mixed-effects models to account for repeated measurements.

  • For nucleolar segregation analysis:

    • Contingency table analysis with chi-square or Fisher's exact test for categorical outcomes .

    • Ordinal logistic regression if categorizing degree of segregation.

• For transcriptional analysis (e.g., rRNA synthesis):

  • For EU signal quantification or Northern blot analysis:

    • Paired and unpaired two-sided Student's t-tests as applied in previous studies .

    • Normalize to appropriate housekeeping genes or total RNA.

    • Consider ANCOVA when controlling for baseline differences between wild-type and UFL1-deficient cells.

• For proteomic identification of ufmylated substrates:

  • False discovery rate (FDR) control for multiple hypothesis testing.

  • Volcano plots displaying both statistical significance and fold change.

  • Enrichment analysis for functional categorization of identified substrates.

• For time-course experiments:

  • Repeated measures ANOVA or mixed-effect models.

  • Survival analysis methods (Kaplan-Meier, Cox proportional hazards) for time-to-event data.

• Data visualization recommendations:

  • Box plots with individual data points rather than bar graphs to show data distribution.

  • Include medians (as shown in Figure 3B of reference ) rather than just means.

  • Use consistent error representation (standard deviation, standard error, or confidence intervals).

Regardless of the specific test, ensure appropriate sample sizes through power analysis, verify test assumptions, and report effect sizes alongside p-values to provide complete statistical information.

How can I integrate findings from in vitro UFL1 studies with cellular and organismal phenotypes?

Integrating findings across different levels of biological organization requires a systematic approach to connect molecular mechanisms to physiological outcomes. Here's a framework for integrating UFL1 research findings:

• Building a hierarchical model of UFL1 function:

  • Start with biochemical characterization of wild-type and mutant UFL1 proteins:

    • E3 ligase activity

    • Protein-protein interactions

    • Substrate specificity

  • Connect these properties to cellular processes:

    • ER homeostasis

    • Ribosome-associated quality control

    • Nucleolar functions and rDNA transcription

    • DNA damage responses

  • Extend to tissue and organismal phenotypes:

    • Tissue-specific expression patterns

    • Developmental roles

    • Stress responses

• Structure-function correlation:

  • Map the effects of specific domain mutations to both enzymatic activities and cellular phenotypes.

  • For example, mutations affecting UFC1 binding should impair all ufmylation-dependent processes, while substrate-binding mutations might show more selective effects.

  • This approach can reveal which molecular functions of UFL1 are critical for specific cellular processes.

• Comparative analysis across models:

  • Compare findings between in vitro, cellular, and in vivo models.

  • Evaluate conservation of UFL1 functions between chicken and mammalian systems.

  • Discrepancies between models can reveal context-dependent regulation or compensatory mechanisms.

• Systems biology approaches:

  • Network analysis to place UFL1 within protein-protein interaction networks.

  • Pathway enrichment analysis of genes/proteins affected by UFL1 deficiency.

  • Multi-omics integration (transcriptomics, proteomics, ufmylome) to build comprehensive models.

• Translational relevance assessment:

  • Connect basic findings to potential biological significance:

    • Developmental roles

    • Stress response mechanisms

    • Cellular quality control pathways

• Visualization strategies for multi-level data integration:

  • Create conceptual models that illustrate connections between molecular events and cellular outcomes.

  • Develop hierarchical heatmaps showing effects of different UFL1 mutations across multiple assays.

  • Use pathway diagrams to contextualize UFL1's role within broader cellular processes.

This integrative approach can generate testable hypotheses about how specific molecular properties of UFL1 contribute to cellular and organismal phenotypes, providing a comprehensive understanding of UFL1 biology.

How can I develop specific inhibitors or modulators of UFL1 activity for research applications?

Developing specific modulators of UFL1 activity requires a systematic approach spanning computational, biochemical, and cellular validation strategies:

• Structural characterization approaches:

  • Determine the three-dimensional structure of UFL1 using X-ray crystallography, cryo-EM, or NMR spectroscopy.

  • In the absence of complete structural data, use homology modeling and molecular dynamics simulations.

  • Identify druggable pockets, particularly at protein-protein interaction surfaces between UFL1 and UFM1, UFC1, or substrates.

• High-throughput screening strategies:

  • Develop robust biochemical assays amenable to high-throughput screening:

    • FRET-based assays monitoring UFM1 transfer.

    • AlphaScreen for detecting protein-protein interactions.

    • Cellular reporter systems (e.g., luciferase-based) reflecting UFL1 activity.

  • Screen compound libraries, focusing on:

    • ATP-competitive compounds targeting E1-E2-E3 cascade.

    • Protein-protein interaction disruptors.

    • Allosteric modulators affecting UFL1 conformation.

• Rational design approaches:

  • Design peptide inhibitors mimicking key interaction motifs:

    • UFC1-binding regions of UFL1.

    • UFBP1-binding surfaces.

    • Substrate recognition elements.

  • Develop structure-based modifications to enhance specificity and cell permeability.

• Validation hierarchies:

  • In vitro biochemical confirmation of target engagement and activity modulation.

  • Cellular validation using ufmylation assays and functional readouts.

  • Selectivity profiling against other E3 ligases and ubiquitin-like modification pathways.

  • Assessment of effects on known UFL1-dependent processes (ER-RQC, nucleolar segregation).

• Tool compound development:

  • Optimize lead compounds for:

    • Potency (sub-micromolar IC₅₀ values).

    • Selectivity (minimal off-target effects on other E3 ligases).

    • Cell permeability and stability.

    • Suitable pharmacokinetic properties for in vivo studies.

  • Develop both inhibitors and activators to provide complementary research tools.

• Application strategies:

  • Use these tools to probe specific aspects of UFL1 biology:

    • Acute vs. chronic inhibition effects.

    • Tissue-specific functions.

    • Context-dependent roles (stress conditions vs. normal growth).

This approach can yield valuable chemical probes to dissect UFL1 functions with temporal and spatial precision beyond what genetic approaches allow.

What are the emerging techniques for mapping the complete chicken ufmylome?

Mapping the complete chicken ufmylome (the set of all proteins modified by UFM1) requires specialized techniques that can identify and characterize these modifications at a proteome-wide scale:

• MS-based identification strategies:

  • Proximity-dependent biotin identification (BioID) or TurboID with UFM1 as bait to identify the ufmylation machinery and substrates.

  • Stable isotope labeling with amino acids in cell culture (SILAC) comparing wild-type and UFL1-deficient cells to identify differentially modified proteins.

  • Diglycine remnant antibody-based enrichment adapted for UFM1, which also leaves a diglycine motif after trypsin digestion of modified proteins.

• Engineered UFM1 approaches:

  • Express biotin-tagged or His-tagged UFM1 in chicken cells or transgenic models, similar to the FLAGHis-Ufm1 transgenic mouse model described .

  • Use tandem affinity purification to isolate ufmylated proteins with high specificity.

  • Combine with CRISPR/Cas9 genome editing to introduce tags at the endogenous UFM1 locus.

• Substrate trapping methods:

  • Express the UFM1-G83A mutant (equivalent to G82A in humans) which forms stable conjugates resistant to deconjugating enzymes .

  • Use this approach to trap and identify transient ufmylation targets that might be missed in steady-state analyses.

• Tissue-specific and subcellular profiling:

  • Fractionate chicken tissues and cells to identify compartment-specific ufmylation targets.

  • Focus on liver and lung tissues, which showed abundant ufmylation in mammalian studies .

  • Isolate ER membranes specifically, given the enrichment of the ufmylation machinery at the ER .

• Bioinformatic prediction and validation:

  • Develop machine learning algorithms to predict potential ufmylation sites based on known substrates.

  • Combine with evolutionary conservation analysis between chicken and mammalian systems.

  • Validate predicted sites through targeted mutagenesis and in vitro ufmylation assays.

• Integration with other -omics approaches:

  • Correlate the ufmylome with transcriptome, proteome, and interactome data.

  • Analyze changes in the ufmylome under various stress conditions (ER stress, DNA damage).

  • Use network analysis to identify functional clusters of ufmylated proteins.

These complementary approaches can provide a comprehensive map of the chicken ufmylome, revealing both conserved and potentially avian-specific targets of this post-translational modification system.

How can CRISPR-based techniques advance our understanding of UFL1 function in avian systems?

CRISPR-based technologies offer powerful approaches to investigate UFL1 function in avian systems with unprecedented precision and versatility:

• Genome editing applications:

  • Generate complete UFL1 knockout lines in chicken cell models (DF-1, DT40) to study essential functions.

  • Create knock-in models expressing tagged versions of UFL1 (FLAG, GFP) at endogenous loci for localization and interaction studies.

  • Introduce specific point mutations to disrupt key functional domains:

    • UFC1-binding regions

    • UFBP1 interaction surfaces

    • Catalytic residues

• Conditional and tissue-specific approaches:

  • Implement CRISPR interference (CRISPRi) with dCas9-KRAB to achieve tunable repression of UFL1 expression.

  • Develop CRISPR activation (CRISPRa) systems with dCas9-VP64 or dCas9-SAM to upregulate UFL1 in specific contexts.

  • Use tissue-specific or inducible promoters to drive Cas9 expression for spatiotemporal control of UFL1 disruption.

• High-throughput functional genomics:

  • Conduct CRISPR screens in chicken cells to identify genes that interact with UFL1:

    • Synthetic lethal screens in UFL1-depleted backgrounds to find compensatory pathways.

    • Suppressor screens to identify genes that rescue UFL1 deficiency phenotypes.

    • Pathways enrichment analysis from screen results to map the functional context of UFL1.

• In vivo applications in chicken embryos:

  • Use in ovo electroporation to deliver CRISPR components targeting UFL1 during development.

  • Implement mosaic analysis to study cell-autonomous effects of UFL1 disruption.

  • Combine with lineage tracing to assess developmental consequences of UFL1 deficiency.

• Base and prime editing applications:

  • Utilize cytosine or adenine base editors to introduce specific mutations without double-strand breaks.

  • Apply prime editing for precise modifications to study structure-function relationships in UFL1.

  • These approaches minimize off-target effects and can be used to model specific variants.

• Visualization and dynamics studies:

  • Implement CRISPR imaging with dCas9-fluorescent protein fusions to track UFL1 locus dynamics.

  • Use split-GFP approaches to visualize UFL1 interactions with partners in live cells.

  • Combine with optogenetic tools for light-controlled modulation of UFL1 activity.

These CRISPR-based approaches provide a versatile toolkit to dissect UFL1 function across multiple biological scales, from molecular interactions to developmental roles, advancing our understanding of this important E3 ligase in avian systems.

How does chicken UFL1 compare structurally and functionally to its mammalian counterparts?

A comprehensive comparative analysis of chicken UFL1 with its mammalian counterparts reveals both conservation and potential divergence in structure and function:

• Sequence conservation analysis:

  • Chicken UFL1 shares approximately 85-90% amino acid sequence identity with human and mouse orthologues, suggesting high functional conservation.

  • The highest conservation typically occurs in functional domains:

    • UFC1-binding regions

    • UFBP1 interaction surfaces

    • Substrate recognition elements

  • C-terminal regions often show greater divergence, potentially reflecting species-specific regulatory mechanisms.

• Domain architecture comparison:

  • The core catalytic domains are highly conserved across species, indicating preservation of the fundamental ufmylation mechanism.

  • Differences in flanking regions may influence regulation, localization, or specific protein-protein interactions.

  • Comparative modeling can highlight structural variations that might impact substrate specificity or enzymatic efficiency.

• Expression pattern differences:

  • In mammals, UFL1 expression is abundant in secretory tissues, particularly liver and lungs .

  • Avian-specific expression patterns may reflect adaptations to bird physiology, such as air sac systems and unique metabolic demands.

  • Developmental expression timing may differ, potentially relating to the distinct developmental programs of avian embryos.

• Cellular localization:

  • Both mammalian and avian UFL1 likely localize predominantly to the endoplasmic reticulum, consistent with its role in ER-associated processes .

  • Subtle differences in targeting sequences or interaction partners might influence subcellular distribution.

  • The nucleolar association during DNA damage responses may show species-specific regulation .

• Functional conservation in cellular processes:

  • Core functions in ER homeostasis, ribosome-associated quality control, and protein folding are likely conserved across vertebrates.

  • The role in nucleolar segregation and rDNA damage response demonstrated in mammalian cells may have parallel functions in avian systems.

  • The effects of UFL1 deficiency on rRNA transcription observed in human cells suggest similar regulatory mechanisms may exist in birds.

• Substrate specificity:

  • Known mammalian substrates like C20orf116/UFBP1 and RPL26 likely have orthologous targets in chicken.

  • Species-specific substrates may exist, potentially relating to unique aspects of avian physiology or stress responses.

Comparative studies between chicken and mammalian UFL1 can provide insights into evolutionarily conserved functions while potentially revealing avian-specific adaptations of the ufmylation system.

What can we learn from comparative studies of UFL1 across different avian species?

Comparative studies of UFL1 across diverse avian species can provide valuable insights into evolutionary patterns, functional conservation, and adaptive specialization:

• Evolutionary rate analysis:

  • Examine selection pressures on UFL1 across the avian phylogeny:

    • Regions under purifying selection likely represent core functional domains.

    • Accelerated evolution might indicate adaptation to species-specific requirements.

  • Compare evolutionary rates between UFL1 and other components of the ufmylation machinery (UBA5, UFC1, UFBP1) to identify co-evolutionary patterns.

• Correlation with avian life-history traits:

  • Analyze UFL1 sequence variation in relation to:

    • Metabolic rates (slow vs. fast metabolism birds)

    • Lifespan (short-lived vs. long-lived species)

    • Environmental adaptations (extreme habitats, migratory vs. non-migratory)

    • Developmental programs (altricial vs. precocial species)

  • These correlations could reveal how UFL1 functions might be tailored to specific physiological demands.

• Expression pattern comparison:

  • Compare tissue-specific expression profiles across diverse avian taxa:

    • Galliformes (chickens, turkeys) as agricultural models

    • Passeriformes (songbirds) as neurobiological models

    • Ratites (ostriches, emus) representing basal avian lineages

  • Identify conserved expression patterns that might indicate core functions versus divergent patterns suggesting specialized roles.

• Structure-function relationship across avian diversity:

  • Compare UFL1 protein structures between species with different physiological demands:

    • High-altitude adapted birds (bar-headed geese) vs. lowland relatives

    • Diving birds (penguins, cormorants) vs. non-diving relatives

    • Migratory birds with high metabolic demands vs. sedentary species

  • These comparisons can reveal structural adaptations that might enhance UFL1 function under specific physiological conditions.

• Integration with genomic and transcriptomic datasets:

  • Analyze UFL1 in context of whole-genome data from initiatives like the B10K (Bird 10,000 Genomes) project.

  • Examine co-expression networks across species to identify conserved and divergent regulatory relationships.

  • Look for lineage-specific duplication or loss events affecting the ufmylation machinery.

• Experimental testing of species-specific variants:

  • Express UFL1 from different avian species in cellular models.

  • Test for functional differences in activity, substrate specificity, or stress responses.

  • Perform domain swapping between species to identify regions responsible for functional divergence.

These comparative approaches can reveal how UFL1 has evolved across the avian radiation, providing insights into both fundamental aspects of protein quality control and species-specific adaptations in this diverse vertebrate lineage.

How does the UFM1 pathway in avian systems adapt to unique physiological stresses?

The UFM1 pathway in avian systems likely exhibits adaptations to address the unique physiological stresses birds encounter, reflecting their distinctive metabolism, thermoregulation, and environmental challenges:

• Adaptations to high metabolic demands:

  • Birds have exceptionally high metabolic rates, which increases protein synthesis demands and potential for proteotoxic stress.

  • The UFL1-mediated ufmylation pathway may show enhanced efficiency or regulation in avian systems to cope with elevated protein turnover.

  • Comparative analysis might reveal avian-specific regulatory elements in UFL1 promoters or post-translational modifications that enable rapid modulation of activity in response to metabolic fluctuations.

• Thermoregulatory stress responses:

  • Birds maintain higher body temperatures (40-42°C) than mammals.

  • This thermal environment likely requires specialized protein quality control systems to prevent misfolding and aggregation.

  • The UFL1/UFM1 pathway may show adaptations to function optimally at these elevated temperatures, potentially through enhanced thermostability of the enzymes or temperature-sensitive regulatory mechanisms.

• Respiratory system specializations:

  • The avian respiratory system, with air sacs and unidirectional airflow, creates unique oxidative stress patterns.

  • Since UFL1 is highly expressed in mammalian lungs , its role may be particularly important in avian respiratory tissues.

  • The ufmylation pathway might have evolved specialized functions to protect against oxidative damage in the avian respiratory epithelium.

• Flight-related adaptations:

  • The extreme energy demands of flight create significant physiological stress.

  • UFL1-mediated quality control may be particularly critical in flight muscles, which require precise protein homeostasis.

  • Comparison between flying and flightless birds might reveal adaptations in the ufmylation pathway related to this specialized locomotion.

• Developmental constraints:

  • Rapid embryonic development within eggs creates unique proteostatic challenges.

  • The UFM1 pathway may show developmental stage-specific regulation in avian embryos.

  • The role of UFL1 in nucleolar functions and rDNA transcription could be particularly important during the accelerated growth phases of avian development.

• Immune system specializations:

  • Birds possess a distinct immune system architecture (lacking lymph nodes, having specialized organs like the bursa of Fabricius).

  • UFL1-mediated processes might play roles in avian-specific immune responses, particularly in protein quality control during antibody production.

• Seasonal adaptations:

  • Many birds undergo dramatic physiological changes during migration, breeding, or molt.

  • The ufmylation pathway may show seasonal regulation to accommodate these cyclical demands.

  • Comparative analysis of UFL1 activity across these physiological states could reveal dynamic regulation mechanisms.

Understanding these avian-specific adaptations of the UFM1 pathway can provide insights not only into bird physiology but also into the general principles of how protein modification systems evolve to meet specialized physiological demands.

What are the most significant unanswered questions about UFL1 in avian systems?

Despite progress in understanding UFL1 and the ufmylation pathway, several critical questions remain unexplored, particularly in avian systems:

• Substrate specificity determinants:

  • What structural features of UFL1 determine substrate recognition in avian cells?

  • How do these compare to mammalian systems, and are there avian-specific substrates?

  • What is the complete ufmylome in chicken cells and how does it respond to different stressors?

• Developmental roles:

  • Is UFL1 essential for avian embryonic development?

  • Are there stage-specific functions during key developmental transitions?

  • How is the UFL1/UFM1 pathway regulated during the rapid development of chicken embryos?

• Tissue-specific functions:

  • What are the tissue-specific expression patterns and functions of UFL1 in diverse avian tissues?

  • Are there specialized roles in avian-specific organs or adaptations (air sacs, bursa of Fabricius)?

  • How do expression patterns correlate with tissue-specific stress responses?

• Regulatory mechanisms:

  • What transcriptional and post-translational mechanisms regulate UFL1 activity in avian cells?

  • How is UFL1 regulated during different physiological states (fasting, egg-laying, migration)?

  • Are there avian-specific regulatory proteins that modulate UFL1 function?

• Pathological implications:

  • What role does UFL1 dysfunction play in avian diseases?

  • Could alterations in the ufmylation pathway contribute to pathologies in poultry or wild birds?

  • Does UFL1 activity affect susceptibility to avian-specific pathogens?

• Evolutionary adaptations:

  • How has the UFL1/UFM1 pathway evolved across the avian phylogeny?

  • Are there lineage-specific adaptations in birds with extreme physiological demands?

  • What can comparative genomics tell us about the evolution of this pathway in vertebrates?

• Integration with other cellular pathways:

  • How does UFL1-mediated ufmylation interact with other post-translational modification systems in avian cells?

  • What is the relationship between the UFM1 pathway and avian-specific metabolic pathways?

  • How does UFL1 function coordinate with other cellular stress response systems?

• Nucleolar functions:

  • Given the role of UFL1 in nucleolar segregation and rDNA transcription in mammalian cells , what parallel functions exist in avian systems?

  • How might these functions relate to the unique nucleolar dynamics in oocytes of egg-laying species?

Addressing these questions will require interdisciplinary approaches combining molecular, cellular, developmental, and evolutionary techniques, ultimately providing a comprehensive understanding of UFL1 biology in avian systems.

What promising future directions exist for UFL1 research in avian models?

Several promising research directions can advance our understanding of UFL1 in avian systems, with potential implications for both basic biology and applied sciences:

• Integrative multi-omics approaches:

  • Combine proteomics, transcriptomics, and genomics to create comprehensive maps of the avian ufmylation system.

  • Develop chicken-specific antibodies and reporter systems to track ufmylation dynamics.

  • Apply spatial transcriptomics and proteomics to understand tissue-specific roles of UFL1.

• Advanced genetic models:

  • Develop conditional UFL1 knockout chicken models using CRISPR/Cas9 technology.

  • Create reporter lines expressing fluorescently tagged UFL1 or UFM1 for real-time imaging.

  • Implement tissue-specific or inducible systems to manipulate UFL1 activity in specific contexts.

• Comparative evolutionary studies:

  • Expand comparative analyses across diverse avian species to identify evolutionary patterns.

  • Investigate UFL1 sequence and function in species with extreme physiological adaptations.

  • Reconstruct the evolutionary history of the UFM1 pathway across vertebrates to understand avian-specific innovations.

• Developmental biology applications:

  • Leverage the accessibility of the chicken embryo to study UFL1's role in development.

  • Investigate potential functions in organogenesis, particularly in tissues where ufmylation is abundant.

  • Explore interactions between the ufmylation pathway and developmental signaling networks.

• Stress response mechanisms:

  • Characterize UFL1's role in avian-specific stress responses, such as heat stress in commercial poultry.

  • Investigate functions during hypoxic stress, relevant to high-altitude adaptation in birds.

  • Examine potential roles in regulating metabolic stress during migration or fasting.

• Agricultural applications:

  • Explore associations between UFL1 variants and production traits in commercial poultry.

  • Investigate potential links to disease resistance or stress tolerance.

  • Develop biomarkers based on ufmylation status to monitor poultry health and welfare.

• Technological innovations:

  • Develop chicken-specific protocols for proximity labeling to identify UFL1 interactors.

  • Implement advanced imaging techniques to visualize ufmylation dynamics in avian cells.

  • Create high-throughput screening platforms to identify modulators of the ufmylation pathway.

• Translational bridging studies:

  • Use insights from avian UFL1 research to inform understanding of mammalian systems.

  • Investigate conserved mechanisms that might be relevant to human diseases.

  • Develop comparative frameworks to identify fundamental versus species-specific aspects of ufmylation.

These research directions capitalize on the unique advantages of avian models while addressing fundamental questions about protein homeostasis, stress responses, and evolutionary adaptation, positioning UFL1 research at the intersection of basic and applied avian biology.

How might insights from UFL1 research in avian systems translate to broader understanding of protein modification pathways?

Research on UFL1 in avian systems has significant potential to enhance our broader understanding of protein modification pathways through several conceptual and methodological contributions:

• Evolutionary insights into modification system diversification:

  • Avian UFL1 research provides a comparative framework to understand how ubiquitin-like modification systems evolved across vertebrate lineages.

  • Differences between avian and mammalian ufmylation can reveal which aspects are fundamental to the pathway versus lineage-specific adaptations.

  • These comparisons help elucidate how novel protein modification systems emerge and specialize during evolution.

• Understanding tissue-specific regulation of modification pathways:

  • The distinctive physiology of avian tissues offers unique contexts to study how modification systems adapt to specialized cellular environments.

  • Comparing UFL1 function across avian tissues can reveal principles of context-dependent regulation applicable to other modification systems.

  • These insights can inform how protein modifications are tailored to the needs of specific cell types or physiological states.

• Stress response adaptation mechanisms:

  • Birds experience distinctive physiological stresses (high body temperature, metabolic demands of flight, etc.).

  • Studying how the ufmylation pathway responds to these avian-specific stresses can reveal general principles of how modification systems are mobilized during cellular stress.

  • These principles may apply to other modification pathways in diverse organisms.

• Integration of modification systems:

  • Research in avian systems can reveal how the ufmylation pathway interacts with other protein modification systems (ubiquitination, SUMOylation, etc.).

  • The avian cellular context may reveal unique crosstalk mechanisms that illuminate broader principles of modification network integration.

  • These interactions provide insights into how cells coordinate multiple modification pathways to maintain proteostasis.

• Developmental regulation of protein modifications:

  • The accessible nature of avian embryos makes them excellent models for studying developmental roles of protein modifications.

  • Understanding how UFL1 functions change during avian development can reveal principles applicable to temporal regulation of other modification systems.

  • These insights connect protein modifications to broader developmental programs across species.

• Methodological innovations:

  • Technical approaches developed for studying UFL1 in avian systems (specific antibodies, activity assays, imaging techniques) can be adapted for other modification systems.

  • Comparative approaches between avian and mammalian systems can identify the most robust and generalizable methods for studying protein modifications.

  • Novel screening approaches in avian cells might identify modulators of modification pathways with broader applicability.

• Therapeutic relevance:

  • Insights from avian UFL1 research may identify conserved functions relevant to human diseases involving protein homeostasis.

  • Understanding how birds regulate protein quality control through ufmylation might suggest novel therapeutic approaches for diseases involving ER stress or protein misfolding.

  • The evolutionary distance between birds and mammals helps distinguish essential pathway functions from species-specific features, clarifying which aspects represent the most promising therapeutic targets.