Recombinant Danio rerio NEDD8-activating enzyme E1 regulatory subunit (nae1), partial

What is Danio rerio NAE1 and what is its function in cellular processes?

NAE1 (NEDD8 Activating Enzyme E1 Subunit 1) functions as the regulatory subunit of the NEDD8-activating enzyme E1, which is essential for the neddylation pathway. In zebrafish, as in mammals, NAE1 forms a heterodimeric complex with UBA3, constituting the E1 enzyme that initiates the neddylation cascade. This process involves the ATP-dependent activation of NEDD8, which is subsequently transferred to E2 conjugating enzymes and ultimately to substrate proteins via E3 ligases .

Functionally, nae1 participates in numerous cellular processes including:

• Regulation of cell cycle progression

• Modulation of signal transduction pathways

• Involvement in developmental processes

• Maintenance of cellular homeostasis during stress conditions

• Regulation of protein degradation via the ubiquitin-proteasome system

The importance of NAE1 is evidenced by studies showing that disruption of neddylation in zebrafish significantly affects the expression of various developmental and homeostatic genes .

How does the neddylation pathway operate in zebrafish compared to mammals?

The neddylation pathway in zebrafish closely resembles that observed in mammals, operating through a three-enzyme cascade:

• Activation: Zebrafish nedd8 is activated by the E1 enzyme complex (nae1/uba3)

• Conjugation: Activated nedd8 is transferred to the E2 enzyme (ubc12)

• Ligation: E3 ligases facilitate the conjugation of nedd8 to target substrate proteins

Experimental evidence demonstrates that zebrafish nedd8 enhances yap1 transcriptional activity similarly to mammalian NEDD8. Promoter luciferase reporter assays confirm that nedd8 overexpression activates yap1-driven promoter activity, while treatment with MLN4924 (an NAE inhibitor) inhibits this activity .

Comparative functional analyses show that overexpression of zebrafish E1 (uba3) and E2 (ubc12) significantly enhances yap1-driven promoter activity, whereas overexpression of the deneddylation enzyme senp8 produces effects similar to MLN4924 treatment .

What experimental models are available to study nae1 function in zebrafish?

Several experimental approaches are available for investigating nae1 function in zebrafish:

These models have revealed that disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes, demonstrating the critical role of the neddylation pathway in Hippo-YAP signaling regulation .

How can recombinant Danio rerio NAE1 be effectively expressed and purified for in vitro studies?

Recombinant Danio rerio NAE1 can be efficiently expressed and purified using the following optimized protocol:

• Expression system selection: While E. coli systems are commonly used for mammalian NAE1 expression , for zebrafish NAE1, both prokaryotic (E. coli) and eukaryotic (insect cell) expression systems are viable options. The choice depends on experimental requirements for post-translational modifications.

• Vector construction:

  • Clone the nae1 cDNA (corresponding to amino acids 1-534 for full-length protein)

  • Insert into an appropriate expression vector containing a His-tag or GST-tag for purification

  • Verify sequence integrity through Sanger sequencing

• Expression conditions:

  • For E. coli: Induce with IPTG (0.1-0.5 mM) at lower temperatures (16-18°C) for 16-20 hours to enhance solubility

  • For insect cells: Use baculovirus expression system with 72-96 hour expression period

• Purification strategy:

  • Lyse cells in buffer containing 10 mM Hepes, 500 mM NaCl, pH 7.4

  • Purify using affinity chromatography (Ni-NTA for His-tagged proteins)

  • Apply size exclusion chromatography to enhance purity (>95%)

  • Stabilize with 5% trehalose prior to lyophilization

• Quality control:

  • Verify purity via SDS-PAGE (aim for >95% purity)

  • Confirm identity by Western blotting using NAE1-specific antibodies

  • Assess functional activity through in vitro neddylation assays

For optimal protein stability, store lyophilized protein at -20°C for up to 12 months, and reconstituted protein at 2-8°C for up to 1 month under sterile conditions .

What are the optimal conditions for assessing NAE1 enzymatic activity in zebrafish systems?

To effectively measure NAE1 enzymatic activity in zebrafish systems, researchers should consider these methodological approaches:

• In vitro neddylation assays:

  • Reaction buffer: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM ATP, 1 mM DTT

  • Components: Recombinant zebrafish nedd8, E1 (nae1/uba3 complex), E2 (ubc12), and substrate protein

  • Detection: Western blotting using anti-nedd8 antibodies to visualize neddylated products

  • Controls: Include reactions without ATP and with the NAE inhibitor MLN4924

• Cellular neddylation assays:

  • Transfect cells with tagged nedd8 and potential substrate proteins

  • Immunoprecipitate the substrate and probe for nedd8 modification

  • Apply denaturing conditions to differentiate covalent nedd8 conjugation from non-covalent interactions

  • Validate with MLN4924 treatment, which should reduce neddylation

• Promoter-reporter assays:

  • Utilize the 8xGTIIC-luciferase reporter system to monitor YAP1 activity

  • Co-transfect with nae1, uba3, and ubc12 to assess the impact on YAP1 transcriptional activity

  • Include appropriate controls: wild-type YAP1, nedd8-ΔGG mutant, and senp8 deneddylase

• Temperature and pH optimization:

  • Optimal temperature range: 25-28°C (physiological for zebrafish)

  • Optimal pH range: 7.2-7.6

• Kinetic analysis:

  • Measure initial reaction rates at varying substrate concentrations

  • Determine Km and Vmax values specific to zebrafish NAE1

  • Compare with mammalian counterparts to identify species-specific differences

Evidence from zebrafish studies demonstrates that overexpression of nedd8, uba3, and ubc12 enhances yap1-driven promoter activity, confirming the functionality of these assay systems .

How can researchers effectively manipulate neddylation in zebrafish models?

Researchers can manipulate neddylation in zebrafish models through several complementary approaches:

• Pharmacological inhibition:

  • MLN4924 (pevonedistat): Specific inhibitor of NAE, effectively blocks the initial step of neddylation

  • Application methods: Add directly to water for embryos/larvae or inject into adult zebrafish

  • Concentration range: 0.1-10 μM, depending on developmental stage and desired effect

  • Monitoring: Assess cullin neddylation status as readout of global neddylation inhibition

• Genetic manipulation:

  • Gene editing: CRISPR/Cas9-mediated knockout or mutation of nae1, uba3, or ubc12

  • Conditional approaches: Use of Cre-loxP or heat-shock inducible systems for temporal control

  • Morpholino knockdown: Transient reduction of nae1 expression during early development

  • Transgenic overexpression: Ectopic expression of wild-type or mutant forms of pathway components

• Manipulation of deneddylation:

  • Overexpression of SENP8/DEN1 deneddylase to accelerate nedd8 removal

  • SENP8 inhibitors to reduce deneddylation and increase nedd8 conjugation

• Substrate-specific approaches:

  • Expression of neddylation-resistant mutants (K→R mutations at neddylation sites)

  • Targeted E3 ligase manipulation to affect specific neddylation substrates

Research shows that disruption of nedd8 in zebrafish results in altered expression of yap1 target genes, including downregulation of yap1-activated genes (ctgf, ccnb1, cdc20, areg, ccna2, aukb, gli2, cyr61, auka) and upregulation of yap1-repressed genes (gadd45a) . These gene expression changes can serve as readouts for effective neddylation manipulation.

How does neddylation regulate the Hippo-YAP signaling pathway in zebrafish development?

Neddylation plays a critical regulatory role in the Hippo-YAP signaling pathway in zebrafish development through several mechanisms:

• Direct neddylation of yap1:

  • Zebrafish yap1 is directly modified by nedd8 conjugation

  • Co-expression of nedd8 and yap1 in cells results in high-molecular-weight bands representing nedd8-conjugated yap1

  • These conjugates disappear when using nedd8-ΔGG, a mutant incapable of covalent binding

  • MLN4924 treatment decreases nedd8-conjugated yap1, while overexpression of E1 (uba3) and E2 (ubc12) increases it

• Functional consequences of yap1 neddylation:

  • Neddylation enhances yap1 transcriptional activity

  • Promoter luciferase reporter assays show that nedd8 overexpression activates yap1-driven promoter activity

  • Conversely, inhibition of neddylation with MLN4924 suppresses yap1-driven promoter activity

• Impact on downstream gene expression:

  • In nedd8-null zebrafish, expression analysis reveals:

    • Downregulation of yap1-activated genes (ctgf, ccnb1, cdc20, areg, ccna2, aukb, gli2, cyr61, auka)

    • Upregulation of yap1-repressed genes (gadd45a)

  • Western blot analysis confirms decreased protein levels of yap1 and yap1 target proteins (ctgf and birc5) in nedd8-null zebrafish brain

• Dual regulatory mechanism:

  • Evidence suggests neddylation regulates Hippo-YAP signaling in two ways:

    • Directly neddylating yap1 to activate YAP-mediated signaling

    • Potentially modifying MST1 (albeit weakly) to inhibit the kinase cascade, indirectly activating yap1

This regulatory relationship is critical for proper development, as disruption of neddylation significantly alters yap1-dependent developmental processes in zebrafish.

What are the pathophysiological consequences of NAE1 deficiency in vertebrate models?

NAE1 deficiency produces diverse pathophysiological consequences in vertebrate models, reflecting the critical role of neddylation in multiple cellular processes:

• Neurological effects:

  • Intellectual disability: Human patients with bi-allelic NAE1 variants display intellectual disability

  • Neurodegeneration: NAE1 deficiency leads to infection-triggered neurodegeneration

  • Cellular basis: NAE1 is required during cellular stress (including infections) to protect against neuronal cell death

• Skeletal abnormalities:

  • Ischiopubic hypoplasia: A rare skeletal feature observed in individuals with NAE1 deficiency

  • Developmental implications: Suggests NAE1's importance in skeletal morphogenesis and bone development

• Immunological dysfunction:

  • Stress-mediated lymphopenia: Patients with NAE1 variants show decreased lymphocyte counts, particularly during infections

  • Mechanistic basis: Decreased NF-κB translocation and impaired lymphocyte response to CD3/CD28 stimulation

• Cellular stress response defects:

  • Increased cell death: Fibroblasts from individuals with NAE1 variants show increased cell death when the proteasome system is stressed with MG132

  • Proteasomal connection: Neddylation is required to restore proteasomal function during stress, and NAE1 deficiency disrupts this process

• Developmental consequences in zebrafish:

  • Disruption of nedd8 in zebrafish affects multiple developmental processes

  • Gene expression analysis shows significant alterations in yap1-activated and yap1-repressed genes across multiple tissues (ovary, brain, and muscle)

These findings highlight NAE1's critical role in maintaining cellular homeostasis during stress and its importance for proper development across multiple organ systems.

How can sensitivity analyses using Data Tables be applied to optimize NAE1 enzyme kinetics studies?

Sensitivity analyses using Data Tables provide powerful tools for optimizing NAE1 enzyme kinetics studies by enabling researchers to systematically explore parameter space and identify critical variables affecting enzyme activity. Here's how to implement this approach:

• 1-D Data Table application for single parameter analysis:

  • Set up a spreadsheet with NAE1 kinetic model (e.g., Michaelis-Menten or more complex models)

  • Create a column listing different values of the parameter of interest (e.g., substrate concentration, ATP concentration, or inhibitor concentration)

  • Use Excel's Data Table function to calculate enzyme activity across this parameter range

  • Example implementation:

    • Column F: Various substrate concentrations (0-500 μM)

    • Column G: Formula referencing the calculated enzyme activity

    • Select range F38:G50, go to Data > What-If Analysis > Data Table

    • Set Column input cell to reference substrate concentration in model

• 2-D Data Table application for interaction analysis:

  • Particularly valuable for examining how two parameters interact to affect NAE1 activity

  • Create a matrix with one parameter varied across rows and another across columns

  • Implement using Excel's Data Table function with both row and column input cells specified

  • Typical applications include:

    • Analyzing NAE1 activity across varying substrate and ATP concentrations

    • Examining MLN4924 inhibition at different enzyme concentrations

    • Optimizing buffer conditions (pH vs. ionic strength)

• Example: Optimizing zebrafish NAE1 reaction conditions:

Values represent relative NAE1 activity (1.00 = optimal conditions)

• Implementation considerations:

  • Use array formulas: Data Tables create array formulas that cannot be partially modified

  • Ensure consistent variable naming and cell references

  • Consider extending the analysis to multiple outputs simultaneously (e.g., Km, Vmax, and kcat/Km)

• Advanced applications:

  • Sensitivity analysis of NAE1 mutations: Compare wild-type vs. mutant kinetic parameters

  • Inhibitor optimization: Identify optimal inhibitor concentrations for experimental use

  • Species comparison: Analyze kinetic differences between zebrafish and human NAE1

This approach allows researchers to rapidly identify optimal experimental conditions, understand parameter sensitivity, and design more efficient and informative NAE1 studies .

What are common pitfalls in neddylation assays using recombinant zebrafish NAE1 and how can they be addressed?

Researchers working with recombinant zebrafish NAE1 in neddylation assays frequently encounter several challenges that can compromise experimental results. Here are common pitfalls and their solutions:

• Protein stability issues:

  • Pitfall: Recombinant NAE1 losing activity during storage or experimental procedures

  • Solution: Store lyophilized protein at -20°C and reconstituted protein at 2-8°C for maximum of 1 month under sterile conditions

  • Recommendation: Add stabilizers like 5% trehalose during buffer formulation

  • Validation: Test activity periodically on a standard substrate to ensure enzyme functionality

• Non-specific nedd8 conjugation:

  • Pitfall: Detecting apparent neddylation that occurs non-enzymatically or post-lysis

  • Solution: Include proper controls such as:

    • ATP-depleted reactions

    • Reactions with nedd8-ΔGG mutant that cannot form covalent conjugates

    • MLN4924-treated samples to inhibit NAE activity

  • Recommendation: Use denaturing conditions during cell lysis to disrupt non-covalent interactions

• E1-E2 transfer efficiency:

  • Pitfall: Poor or inconsistent transfer of nedd8 from E1 to E2

  • Solution: Optimize reaction conditions (pH 7.2-7.6, temperature 25-28°C)

  • Recommendation: Ensure proper protein folding by expressing proteins in eukaryotic systems when possible

  • Validation: Monitor thioester intermediate formation with non-reducing SDS-PAGE

• Species-specific differences:

  • Pitfall: Assuming complete functional equivalence between zebrafish and mammalian neddylation components

  • Solution: Verify compatibility when mixing components from different species

  • Recommendation: Use complete zebrafish systems when studying zebrafish-specific processes

  • Validation: Compare efficiencies using parallel assays with species-matched components

• Substrate identification challenges:

  • Pitfall: Difficulty in confirming physiologically relevant neddylation substrates

  • Solution: Combine multiple approaches:

    • In vitro neddylation with recombinant proteins

    • Cell-based assays with overexpression

    • Verification in nedd8-null zebrafish models

  • Recommendation: Identify specific neddylation sites using mass spectrometry

  • Validation: Mutate putative neddylation sites (K→R) to confirm specificity

• Quantification inaccuracies:

  • Pitfall: Challenges in accurately quantifying neddylation levels

  • Solution: Normalize against total protein using stain-free detection gels

  • Recommendation: Use appropriate housekeeping proteins (β-actin or HSP90) for Western blot normalization

  • Validation: Include known quantities of recombinant neddylated proteins as standards

Addressing these pitfalls will significantly improve the reliability and reproducibility of neddylation assays using recombinant zebrafish NAE1.

How can researchers differentiate between direct and indirect effects of NAE1 manipulation in developmental studies?

Differentiating between direct and indirect effects of NAE1 manipulation presents a significant challenge in developmental studies due to the pleiotropic nature of neddylation. The following methodological approaches can help researchers establish causality:

Research has demonstrated that disruption of nedd8 in zebrafish affects multiple downstream pathways but has particularly strong effects on yap1 signaling, resulting in characteristic gene expression changes. This suggests that while NAE1 has many substrates, certain pathways (like Hippo-YAP) are particularly sensitive to neddylation status .

What are the most informative readouts for assessing NAE1 function in zebrafish models?

To comprehensively assess NAE1 function in zebrafish models, researchers should employ multiple complementary readouts that span molecular, cellular, and organismal levels:

• Molecular readouts:

  • Global neddylation status:

    • Western blot analysis of neddylated cullins (CUL1, CUL3), which serve as primary indicators of neddylation pathway activity

    • Quantitative measurement of free vs. conjugated nedd8 levels

  • Target-specific neddylation:

    • Immunoprecipitation followed by nedd8 immunoblotting for specific substrates like yap1

    • In situ proximity ligation assays to visualize protein-nedd8 interactions in tissues

  • Transcriptional responses:

    • qPCR analysis of yap1 target genes (activated: ctgf, ccnb1, cdc20, areg, ccna2, aukb, gli2, cyr61, auka; repressed: gadd45a)

    • RNA-seq for genome-wide transcriptional profiling and pathway analysis

    • Analysis using the Gene Set Enrichment Analysis (GSEA) tool to identify affected pathways

• Cellular readouts:

  • Cell proliferation and death:

    • BrdU/EdU incorporation to assess proliferation rates

    • TUNEL or Acridine Orange staining to measure apoptosis

    • Particularly informative in tissues like developing brain and hematopoietic organs

  • Cell stress responses:

    • Proteasome activity measurements using fluorogenic substrates

    • Assessment of cellular response to stressors (MG132, heat shock, oxidative stress)

    • Analysis of NF-κB nuclear translocation during immune challenges

  • Subcellular localization:

    • Immunofluorescence analysis of yap1 nuclear accumulation

    • Live imaging of fluorescently tagged proteins in transgenic lines

• Tissue and organismal readouts:

  • Developmental timing:

    • Assessment of key developmental milestones

    • Growth rate and body size measurements

  • Tissue-specific phenotypes:

    • Brain: Neuroanatomical analysis, behavioral testing

    • Immune system: Lymphocyte counts during baseline and infection states

    • Skeletal system: Cartilage and bone development, particularly focusing on ischiopubic regions

  • Functional assays:

    • Immune challenge survival assays

    • Regeneration capacity after tissue injury

    • Stress resistance tests

• Comparative readouts:

  • Rescue experiments:

    • Phenotypic rescue with wild-type vs. mutant NAE1

    • Cross-species rescue (human NAE1 in zebrafish)

  • Drug response patterns:

    • Sensitivity to MLN4924 at different doses and developmental stages

    • Differential responses to various cellular stressors

These multi-level readouts together provide a comprehensive assessment of NAE1 function in zebrafish, enabling researchers to connect molecular mechanisms to physiological outcomes and identify the most critical NAE1-dependent processes in development and homeostasis.

What are emerging applications of NAE1 research in disease modeling and therapeutic development?

NAE1 research is revealing promising applications in disease modeling and therapeutic development, with several emerging directions:

• Neurodevelopmental disorder modeling:

  • Bi-allelic NAE1 variants in humans cause intellectual disability and neurodegeneration

  • Zebrafish nae1 models can recapitulate aspects of these disorders, allowing:

    • High-throughput screening of potential therapeutics

    • Investigation of cellular mechanisms underlying neurodegeneration

    • Testing of neuroprotective strategies targeting the neddylation pathway

• Cancer biology applications:

  • YAP1 dysregulation is implicated in multiple cancers

  • The discovered connection between neddylation and YAP1 signaling provides new therapeutic targets

  • Potential applications include:

    • Development of NAE inhibitors with improved specificity compared to MLN4924

    • Testing combination therapies targeting both neddylation and Hippo pathway components

    • Identification of cancer subtypes particularly dependent on NAE1-YAP1 signaling

• Stress resistance and cellular protection:

  • NAE1 is required during cellular stress to protect against cell death

  • This suggests potential applications in:

    • Neuroprotection during acute brain injuries

    • Enhancement of cellular stress resistance in degenerative conditions

    • Prevention of stress-induced lymphopenia during infections

• Developmental biology insights:

  • The role of neddylation in regulating YAP1 provides new understanding of organ size control mechanisms

  • This opens avenues for:

    • Tissue engineering applications targeting controlled growth

    • Regenerative medicine approaches modulating YAP1 activity through neddylation

    • Developmental disorder treatments targeting specific neddylation substrates

• Precision medicine approaches:

  • Patient-derived zebrafish xenografts could test sensitivity to neddylation inhibitors

  • Analysis of neddylation patterns in patient samples may identify disease subtypes

  • Genetic screening could identify individuals particularly sensitive to NAE-targeting drugs

The unique advantages of zebrafish models for these applications include:

• Optical transparency allowing live imaging of disease processes

• High fecundity supporting large-scale drug screens

• Conservation of neddylation pathway components between zebrafish and humans

• Feasibility of genetic manipulation through CRISPR/Cas9 technology

As researchers continue to elucidate the specific roles of NAE1 in development and disease, these emerging applications are likely to translate into concrete therapeutic approaches.

What technological advances would facilitate more precise manipulation of the neddylation pathway in zebrafish?

Several technological advances would significantly enhance researchers' ability to precisely manipulate the neddylation pathway in zebrafish:

• Advanced genetic engineering tools:

  • Base editing technology: For introducing precise point mutations in nae1 or nedd8 without double-strand breaks

  • Prime editing: To enable scarless introduction of specific mutations in neddylation sites

  • Inducible degron systems: For rapid, reversible depletion of NAE1 protein

  • Split-Cas9 systems: For tissue-specific gene editing of neddylation pathway components

• Improved protein visualization techniques:

  • Engineered nedd8 sensors: Fluorescent proteins that change properties when nedd8 is conjugated to targets

  • Advanced FRET/BRET reporters: To visualize neddylation events in real-time in living zebrafish

  • Expansion microscopy adaptations: For super-resolution imaging of neddylation complexes in zebrafish tissues

  • Photoactivatable nedd8 variants: For spatiotemporal control of neddylation processes

• Substrate-specific manipulation approaches:

  • PROTACs targeting specific neddylated proteins: For selective degradation of individual neddylation substrates

  • Neddylation-resistant substrate libraries: Systematic creation of K→R mutants for major neddylation targets

  • E3 ligase-specific inhibitors: To selectively block neddylation of specific substrate classes

  • Engineered NEDD8 variants: With altered specificity for selective substrate targeting

• High-throughput phenotypic analysis:

  • Automated zebrafish phenotyping platforms: For systematic characterization of neddylation pathway mutants

  • Single-cell transcriptomics integration: To identify cell-specific responses to neddylation manipulation

  • AI-powered image analysis: For detecting subtle phenotypic changes in neddylation pathway mutants

  • Behavioral phenotyping tools: To assess neurological impacts of neddylation alterations

• Chemical biology innovations:

  • Covalent inhibitors specific to zebrafish NAE1: With improved specificity over MLN4924

  • Photo-switchable neddylation inhibitors: For precise temporal and spatial control of inhibition

  • E1-E2 interface disruptors: For more specific pathway inhibition than E1 enzymatic inhibitors

  • Chemical genetics approaches: Using engineered NAE1 variants sensitive to specific inhibitors

• Proteomics advances:

  • Improved mass spectrometry protocols: For comprehensive identification of the zebrafish "neddylome"

  • Quantitative neddylation site mapping: To determine stoichiometry of modification at each site

  • Targeted proteomics assays: For routine monitoring of specific neddylation events

  • Crosslinking mass spectrometry: To map neddylation enzyme-substrate interaction networks

These technological advances would collectively enable unprecedented precision in manipulating and monitoring the neddylation pathway in zebrafish, facilitating deeper insights into its developmental and physiological roles.

How might integrated multi-omics approaches enhance our understanding of NAE1 function in development?

Integrated multi-omics approaches offer powerful strategies to comprehensively map NAE1 function across biological scales, revealing regulatory networks and functional impacts that would remain obscured by single-method approaches:

• Integrated proteomics strategies:

  • Global neddylome profiling: Mass spectrometry identification of all nedd8-modified proteins under various developmental conditions

  • Quantitative site-specific mapping: Determination of exact neddylation sites and their occupancy rates during development

  • Protein interaction networks: Proximity labeling (BioID/TurboID) to map the interactome of NAE1 and neddylation machinery

  • Post-translational modification crosstalk: Analysis of how neddylation interfaces with phosphorylation, ubiquitination, and other modifications

• Transcriptomics integration:

  • Developmental stage-specific RNA-seq: Profiling transcriptional changes in nae1-deficient zebrafish across developmental timepoints

  • Single-cell transcriptomics: Mapping cell type-specific responses to neddylation disruption

  • Nascent RNA sequencing: Distinguishing direct transcriptional effects from secondary responses

  • Alternative splicing analysis: Investigating potential impacts of neddylation on RNA processing

  • Gene Set Enrichment Analysis: Identifying significantly affected pathways and biological processes

• Epigenomic approaches:

  • ChIP-seq for YAP1 binding: Mapping how neddylation affects YAP1 chromatin occupancy

  • ATAC-seq analysis: Determining changes in chromatin accessibility in nae1-deficient zebrafish

  • Histone modification profiling: Investigating potential connections between neddylation and histone-modifying enzymes

  • Chromosome conformation capture: Examining effects on 3D genome organization

• Metabolomics integration:

  • Global metabolite profiling: Identifying metabolic consequences of neddylation disruption

  • Flux analysis: Determining how neddylation affects metabolic pathway dynamics

  • Energy metabolism assessment: Given the role of neddylation in stress responses

• Multi-scale data integration frameworks:

  • Temporal mapping: Constructing developmental trajectories of multi-omics data

  • Network modeling: Building integrated regulatory networks connecting neddylation to downstream processes

  • Causal inference approaches: Distinguishing primary from secondary effects of NAE1 manipulation

  • Comparative analysis: Aligning zebrafish data with human and mouse datasets for evolutionary insights

• Practical implementation strategy:

  • Time course design: Sample collection at key developmental stages (cleavage, gastrulation, somitogenesis, organogenesis)

  • Tissue specificity: Focusing on tissues with known neddylation phenotypes (brain, immune cells, developing skeleton)

  • Perturbation conditions: Include normal development, nae1 deficiency, MLN4924 treatment, and stress conditions

  • Data integration platforms: Utilize computational frameworks specifically designed for multi-omics integration

Evidence from studies already demonstrates the power of this approach, as gene expression analysis in nedd8-null zebrafish revealed significant effects on yap1-regulated genes across multiple tissues , while proteomic analysis has identified mechanisms of NAE1 deficiency in human cells .

The integration of these multi-omics approaches would create a comprehensive map of NAE1 function in development, revealing not only direct neddylation targets but also the cascade of biochemical, transcriptional, and cellular changes that drive the observed developmental phenotypes.

What are the key outstanding questions in understanding NAE1 function in zebrafish models?

Despite significant progress in understanding NAE1 function in zebrafish models, several critical questions remain unanswered, presenting opportunities for future research:

• Substrate specificity and regulation:

  • What determines which proteins become neddylated in zebrafish?

  • How is substrate selection regulated during different developmental stages?

  • Are there zebrafish-specific neddylation targets not found in mammals?

  • What is the complete "neddylome" in zebrafish, and how does it change developmentally?

• Developmental timing and tissue specificity:

  • Why do certain tissues (brain, skeletal elements, immune system) appear particularly sensitive to NAE1 disruption?

  • What are the critical developmental windows requiring NAE1 function?

  • How does neddylation interface with other developmental signaling pathways beyond Hippo-YAP?

  • Are there compensatory mechanisms that activate when neddylation is disrupted?

• Mechanistic understanding:

  • What is the precise molecular mechanism by which neddylation enhances YAP1 activity?

  • Which E3 ligases are responsible for neddylation of different substrates in zebrafish?

  • How does lysine 159 neddylation specifically affect YAP1 function?

  • What determines the balance between neddylation and deneddylation in different cellular contexts?

• Stress response and cellular protection:

  • How does neddylation protect cells during stress conditions?

  • What is the molecular basis for the increased cell death observed in NAE1-deficient cells under stress?

  • How does neddylation interface with the unfolded protein response and other stress pathways?

  • Can enhancement of neddylation provide therapeutic protection against certain stressors?

• Evolutionary considerations:

  • How conserved are neddylation targets between zebrafish and mammals?

  • Has the neddylation pathway acquired novel functions in teleost fish?

  • What can comparative studies across species tell us about the evolution of this post-translational modification?

  • Are there differences in neddylation regulation between zebrafish and mammals that affect model translation?

• Technological challenges:

  • How can we achieve temporal and spatial control of neddylation in specific zebrafish tissues?

  • What are the most effective methods to visualize neddylation events in living zebrafish?

  • How can we distinguish between direct and indirect effects of neddylation disruption?

  • What biomarkers can reliably indicate neddylation pathway activity in zebrafish models?

Addressing these questions will require integrated approaches combining genetic, biochemical, and computational methods, but would significantly advance our understanding of this critical post-translational modification in vertebrate development and disease.

How does the current understanding of NAE1 in zebrafish translate to human development and disease?

The current understanding of NAE1 in zebrafish provides valuable insights into human development and disease, with several translational implications:

• Conserved developmental functions:

  • The neddylation pathway components and core mechanisms are highly conserved between zebrafish and humans

  • Zebrafish studies show neddylation regulates YAP1 signaling, a pathway critical for human development

  • Similar to zebrafish findings, human NAE1 deficiency affects multiple organ systems, including the brain and skeletal elements

  • This conservation supports zebrafish as a relevant model for human NAE1-related disorders

• Pathological insights from human NAE1 variants:

  • Bi-allelic variants in human NAE1 cause a syndrome characterized by:

    • Intellectual disability

    • Ischiopubic hypoplasia

    • Stress-mediated lymphopenia

    • Neurodegeneration

  • These features align with zebrafish neddylation pathway disruption phenotypes

  • Zebrafish models can be used to functionally validate human NAE1 variants and understand their pathogenicity

• Mechanistic parallels in cellular stress responses:

  • Both human and zebrafish studies demonstrate that NAE1 is critical during cellular stress

  • NAE1-deficient cells from human patients show increased death when the proteasome is stressed with MG132

  • Similarly, zebrafish studies show neddylation disruption affects stress-responsive pathways

  • This suggests conserved cytoprotective functions of neddylation across species

• Therapeutic implications:

  • NAE inhibitors like MLN4924 have similar effects in zebrafish and human cells

  • Drug development insights from zebrafish models may translate to human applications

  • The identified connection between neddylation and YAP1 signaling provides potential therapeutic targets for human diseases involving YAP1 dysregulation

  • Understanding NAE1's role in cellular protection may lead to neuroprotective strategies for human conditions

• Translational research framework:

  • Zebrafish findings can inform focused studies in mammalian models and human cells

  • Developmental processes affected by nae1 disruption in zebrafish can guide investigations in human developmental disorders

  • Biomarkers of neddylation pathway activity identified in zebrafish may be applicable to human diagnostics

  • Genetic screening in zebrafish can identify modifiers of NAE1-related phenotypes relevant to human disease variability

The bidirectional flow of information between zebrafish and human studies has already proven valuable, with zebrafish research revealing molecular mechanisms (like YAP1 neddylation) that inform human disease understanding, while human genetic findings (NAE1 variants) stimulate targeted zebrafish studies to elucidate pathophysiology. This reciprocal relationship positions zebrafish NAE1 research as an important component of translational science in this field.

What are the optimal protocols for generating and validating nae1 knockout or knockdown zebrafish models?

The following comprehensive protocols provide guidance for generating and validating nae1 knockout or knockdown zebrafish models with high specificity and efficiency:

CRISPR/Cas9-Mediated nae1 Knockout

• gRNA design and validation:

  • Design 3-4 gRNAs targeting early exons of nae1 using tools like CHOPCHOP or CRISPRscan

  • Prioritize gRNAs with high predicted efficiency and low off-target potential

  • Validate gRNA efficiency using in vitro cleavage assays with purified Cas9 protein

  • Recommended target: Exon 2-3 to disrupt the majority of functional domains

• Microinjection procedure:

  • Prepare injection mix: 300 ng/μL Cas9 protein, 100 ng/μL gRNA, 0.05% phenol red

  • Inject 1-2 nL into one-cell stage embryos

  • Include control injections: Cas9 only, unrelated gRNA, and uninjected controls

  • Maintain at 28.5°C in E3 medium supplemented with methylene blue

• Founder (F0) screening:

  • At 24-48 hpf, collect 8-10 injected embryos for DNA extraction

  • Perform T7 endonuclease I assay or direct sequencing to confirm mutagenesis

  • Raise remaining embryos to adulthood

  • At 3-4 months, outcross F0 fish to wild-type and screen offspring for germline transmission

• Establishing stable lines:

  • Identify F1 carriers by fin clip genotyping

  • Sequence identified mutations to determine nature of indels

  • Prioritize frameshift mutations that introduce early stop codons

  • Analyze protein prediction to confirm loss of functional domains

  • Intercross heterozygous F1 carriers to generate homozygous F2 embryos

Morpholino Knockdown Approach

• Morpholino design:

  • Design translation-blocking morpholino targeting nae1 start codon region

  • Design splice-blocking morpholino targeting exon-intron boundaries

  • Consider designing morpholinos against uba3 as alternative approach

  • Include 5-base mismatch control morpholinos

• Morpholino delivery:

  • Prepare working dilutions of 0.25-1.0 mM in nuclease-free water with 0.05% phenol red

  • Inject 1-2 nL into one-cell stage embryos

  • Establish dose-response relationship by testing multiple concentrations

  • Include standard control morpholino injections at equivalent doses

• Essential controls:

  • p53 co-injection to minimize off-target apoptosis

  • Rescue experiments with nae1 mRNA lacking morpholino binding site

  • Comparison with phenotypes observed in established genetic models

  • Replicate key findings using CRISPR/Cas9 F0 "crispants"

Validation Methods for Both Approaches

• Molecular validation:

  • mRNA expression: qRT-PCR analysis of nae1 transcript levels using primers spanning multiple exons

  • Protein expression: Western blot analysis using validated NAE1 antibodies

  • Nonsense-mediated decay: Analysis of potential compensatory upregulation of related genes

  • Neddylation activity: Assessment of global neddylation by cullin immunoblotting

• Functional validation:

  • YAP1 signaling: Expression analysis of YAP1 target genes (ctgf, ccnb1, cdc20, areg, ccna2)

  • Promoter activity: 8xGTIIC-luciferase reporter assays in isolated cells from mutants

  • Stress response: Evaluation of cellular responses to proteasome inhibition

  • Target neddylation: Assessment of YAP1 neddylation status

• Phenotypic validation:

  • Development monitoring: Morphological assessment throughout embryonic and larval stages

  • Tissue-specific analysis: Focus on brain, skeletal elements, and immune system

  • Comparison with reference phenotypes: Cross-reference with published phenotypes from NAE1 deficiency

  • Rescue experiments: Attempt phenotypic rescue with wild-type nae1 mRNA or human NAE1

• Off-target analysis:

  • Whole genome sequencing: For CRISPR-generated lines to identify potential off-target mutations

  • Cross-method validation: Compare CRISPR and morpholino phenotypes

  • Multiple guide validation: Generate independent lines using different gRNAs

  • Allelic series: Analyze multiple alleles of varying severity

Following these protocols will ensure the generation of reliable nae1 loss-of-function zebrafish models that can be confidently used to investigate the role of this gene in development and disease.

What are the key considerations for experimental design when studying neddylation pathway interactions?

Designing rigorous experiments to study neddylation pathway interactions requires careful consideration of multiple factors to ensure valid and reproducible results:

• Control strategy development:

  • Genetic controls:

    • Include wild-type siblings from the same clutch as nae1 mutants

    • Generate and test multiple independent mutant alleles

    • Use heterozygous carriers to assess dose-dependent effects

    • Consider creating "rescue lines" expressing wild-type nae1

  • Pharmacological controls:

    • Include vehicle controls matched to MLN4924 solvent

    • Implement dose-response curves rather than single concentrations

    • Use chemically distinct NAE inhibitors to confirm specificity

    • Test time-dependent effects with treatment time courses

  • Experimental validation controls:

    • Use nedd8-ΔGG mutant (unable to conjugate) as negative control

    • Include E1 (uba3) and E2 (ubc12) overexpression as positive controls

    • Test senp8 (deneddylase) overexpression as pathway antagonist

• Temporal considerations:

  • Developmental timing:

    • Map pathway activity across developmental stages

    • Determine critical windows for neddylation function

    • Consider potential maternal contribution of nae1 mRNA

    • Design time-course experiments spanning key developmental events

  • Signal dynamics:

    • Implement pulse-chase experiments to assess neddylation turnover rates

    • Measure acute vs. chronic responses to pathway perturbation

    • Consider circadian and other temporal variations in pathway activity

• Pathway interaction analysis:

  • Epistasis testing:

    • Create double mutants between nae1 and interacting pathway components

    • Use genetic or pharmacological manipulation of upstream/downstream factors

    • Determine whether phenotypes are additive, synergistic, or epistatic

    • Example: Test interactions between neddylation and Hippo pathway components

  • Multi-pathway considerations:

    • Assess effects on parallel pathways (e.g., ubiquitination, sumoylation)

    • Test for compensatory activation of alternative pathways

    • Consider potential interference from overlapping enzymatic machinery

    • Evaluate stress-specific pathway interactions

• Technical and analytical rigor:

  • Replication strategy:

    • Implement biological replicates (different clutches or animals)

    • Include technical replicates for experimental procedures

    • Calculate appropriate sample sizes based on expected effect sizes

    • Pre-register experimental designs and analysis plans

  • Quantification approaches:

    • Use automated image analysis for phenotypic quantification

    • Implement blinded scoring for subjective measurements

    • Normalize neddylation measurements to total protein levels

    • Apply appropriate statistical tests with correction for multiple comparisons

  • Methodology validation:

    • Validate antibody specificity with knockout controls

    • Confirm siRNA/morpholino knockdown efficiency

    • Test multiple primer pairs for qPCR validation

    • Validate expression constructs in appropriate cell systems

• Experimental system selection:

  • In vivo vs. in vitro approaches:

    • Link cell-based findings to whole-organism phenotypes

    • Validate zebrafish findings in mammalian systems when possible

    • Consider primary cell cultures from zebrafish tissues

    • Determine appropriate developmental stages for tissue isolation

  • Cell type considerations:

    • Select cell types relevant to observed phenotypes

    • Consider tissue-specific differences in neddylation pathway

    • Use fluorescent reporters for cell type-specific analyses

    • Implement cell type-specific Cre lines for conditional approaches

These experimental design considerations will strengthen the validity and impact of research on neddylation pathway interactions, ensuring that findings contribute meaningfully to the understanding of this critical post-translational modification system.

What are key antibodies, reagents, and research tools for studying NAE1 and neddylation in zebrafish?

The following comprehensive list provides essential antibodies, reagents, and research tools optimized for studying NAE1 and neddylation in zebrafish models:

Antibodies and Immunological Reagents

• NAE1-specific antibodies:

  • Anti-NAE1 antibody (Novus Biologicals) - validated for Western blotting at 1:1,000 dilution

  • Anti-NAE1/APPBP1 antibody (Cell Signaling Technology) - for immunofluorescence and IP

  • Anti-His tag antibody - for detection of recombinant His-tagged NAE1 proteins

• Neddylation pathway antibodies:

  • Anti-NEDD8 antibody (Cell Signaling Technology) - detects free and conjugated NEDD8

  • Anti-UBA3 antibody - for detection of the catalytic E1 subunit

  • Anti-UBC12 antibody - for detection of the E2 conjugating enzyme

  • Anti-SENP8/DEN1 antibody - for detecting the major deneddylase

• Substrate and target antibodies:

  • Anti-Cullin 1 antibody - validated at 1:1,000 dilution for detecting neddylated and non-neddylated forms

  • Anti-Cullin 3 antibody - validated at 1:1,000 dilution

  • Anti-YAP1 antibody - for detecting total and potentially neddylated YAP1

  • Anti-CTGF and Anti-BIRC5 antibodies - for YAP1 target validation

• Control and normalization antibodies:

  • Anti-β-actin antibody - validated at 1:1,000 dilution for normalization

  • Anti-HSP90 antibody - alternative for normalization at 1:1,000 dilution

  • Anti-GAPDH antibody - for metabolically active tissues

Chemical Tools and Reagents

• Neddylation pathway modulators:

  • MLN4924 (pevonedistat) - specific inhibitor of NAE

  • Ubiquitin-like modifier activating enzyme (UAE) inhibitors - as controls

  • MG132 - proteasome inhibitor for testing stress responses

  • Cycloheximide - for protein stability/turnover studies

• Recombinant proteins:

  • Recombinant zebrafish NAE1 protein (His-tagged) - for in vitro assays

  • Recombinant zebrafish NEDD8 protein

  • Recombinant zebrafish UBA3 and UBC12 proteins

  • NEDD8-ΔGG mutant protein - critical negative control

• Buffer systems:

  • Zebrafish lysis buffer: 10 mM Hepes, 500 mM NaCl, pH 7.4 with protease inhibitors

  • Neddylation reaction buffer: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM ATP, 1 mM DTT

  • Trehalose-containing buffer (5%) for protein stabilization

Genetic Tools and Resources

• Expression constructs:

  • pLenti CMV Puro vector with nae1 cDNA for rescue experiments

  • 8xGTIIC-luciferase reporter for YAP1 activity assays

  • Expression vectors for zebrafish nedd8, nae1, uba3, ubc12, and senp8

  • NEDD8-ΔGG mutant expression constructs

• Gene editing tools:

  • Validated CRISPR/Cas9 gRNA sequences targeting zebrafish nae1

  • Morpholino antisense oligonucleotides for nae1 knockdown

  • Validated PCR primers for genotyping nae1 mutants

  • shRNA constructs targeting nae1 for knockdown studies

• Transgenic lines:

  • Tg(8xGTIIC:GFP) - reporter line for in vivo YAP1 activity

  • Tissue-specific Gal4 driver lines for targeted manipulation

  • Heat-shock inducible nae1 rescue lines

  • Fluorescent-tagged nedd8 transgenic lines for live imaging

Analytical Tools and Software

• Protein analysis tools:

  • Stain-free detection gels (Bio-Rad) for total protein normalization

  • Mass spectrometry protocols optimized for neddylation site identification

  • Proximity ligation assay kits for in situ detection of neddylation

• Gene expression analysis:

  • Validated qPCR primer sets for yap1 target genes (ctgf, ccnb1, cdc20, areg, ccna2, aukb, gli2, cyr61, auka, gadd45a)

  • Gene Set Enrichment Analysis (GSEA) tool for pathway analysis

  • ClusterProfiler R package for GO term analysis

• Data analysis software:

  • ImageJ with custom macros for quantifying Western blot band intensities

  • Excel Data Table function templates for sensitivity analyses

  • R scripts for statistical analysis of neddylation pathway data