Recombinant Danio rerio Probable E3 ubiquitin-protein ligase RNF144A-B (rnf144ab)

What is RNF144A-B and what structural domains characterize its function?

RNF144A-B contains the characteristic RING1-IBR-RING2 (RBR) domain and a single transmembrane domain that are critical for its E3 ubiquitin ligase activity. The full-length protein (293 amino acids in Danio rerio) includes multiple conserved domains that enable substrate recognition and ubiquitination functions . Structurally, RNF144A-B belongs to the family of RBR E3 ligases, which function through a unique RING/HECT hybrid mechanism. The protein's active site contains critical cysteine residues (particularly C20/C23 in RING1 and C198 in RING2) that are essential for the ubiquitin transfer process during substrate targeting . When working with recombinant forms, researchers should verify the integrity of these domains to ensure proper enzymatic function.

What basic experimental approaches can verify recombinant RNF144A-B activity?

To verify recombinant RNF144A-B activity, researchers should implement a multi-step approach:

• In vitro ubiquitination assays: Using purified components (E1, E2 enzymes, ubiquitin, ATP, and the recombinant RNF144A-B), monitor the formation of polyubiquitin chains via Western blotting.

• Substrate-specific assays: Confirm activity using known substrates such as DNA-PKcs, which has been established as a target of RNF144A .

• Mutational analysis: Compare wild-type protein activity with catalytically inactive mutants (C20A/C23A or C198A) as negative controls to confirm that observed ubiquitination is specifically due to RNF144A-B activity .

• Subcellular localization: Verify proper localization to cytoplasmic vesicles and plasma membrane, which is characteristic of functional RNF144A proteins .

These approaches provide complementary evidence of functional activity and should be conducted before proceeding to more complex experimental applications.

What are the optimal conditions for storage and reconstitution of recombinant RNF144A-B?

For optimal handling of recombinant RNF144A-B:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be maintained at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Create small aliquots and store at -20°C/-80°C

The protein is supplied in Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain stability during storage . Researchers should monitor protein activity after reconstitution by performing activity assays as described in question 1.3.

How should researchers design ubiquitination experiments using recombinant RNF144A-B?

When designing ubiquitination experiments with RNF144A-B, researchers should follow this methodological approach:

• Component preparation:

  • Purify recombinant RNF144A-B to >90% purity (verified by SDS-PAGE)

  • Include essential components: E1 enzyme, appropriate E2 conjugating enzyme, ubiquitin, ATP, and buffer system

• Reaction setup:

  • Include appropriate controls:

    • No-ATP control

    • No-E1 control

    • Catalytically inactive RNF144A-B mutant (C20A/C23A or C198A)

  • Time course analysis (15-60 minutes) to capture reaction kinetics

• Detection methods:

  • Western blotting with anti-ubiquitin antibodies

  • Substrate-specific antibodies to detect shifts in molecular weight

  • Mass spectrometry to identify ubiquitination sites

• Validation in cellular systems:

  • Transfect wild-type and mutant forms into appropriate cell lines

  • Compare ubiquitination levels using immunoprecipitation followed by ubiquitin detection

This comprehensive approach ensures reliable assessment of RNF144A-B E3 ligase activity while controlling for experimental artifacts.

What expression systems are recommended for producing functional zebrafish RNF144A-B?

Based on the available data, the following expression systems are recommended:

Bacterial expression (E. coli):

• Successfully used for producing full-length (1-293aa) His-tagged RNF144A-B

• Advantages: High yield, cost-effective, established protocols

• Limitations: May lack post-translational modifications; proper folding must be verified

Alternative expression systems to consider:

• Insect cells: Better for proteins requiring complex folding and post-translational modifications

• Mammalian cells: Ideal for functional studies where authentic processing is critical

• Cell-free systems: Useful for rapid screening and proteins toxic to host cells

When using any expression system, researchers should:

• Verify protein folding through circular dichroism or limited proteolysis

• Confirm enzymatic activity using in vitro ubiquitination assays

• Assess membrane localization if studying transmembrane domain function

The choice of expression system should align with experimental goals—E. coli is sufficient for basic biochemical characterization, while more complex systems may be necessary for studies of protein-protein interactions or membrane localization.

How does RNF144A-B function in DNA damage response pathways?

RNF144A (the mammalian homolog) plays a critical role in regulating DNA-PKcs during DNA damage response:

• Induction mechanism: DNA damage induces RNF144A expression in a p53-dependent manner, establishing it as part of the cellular response to genotoxic stress .

• Subcellular targeting: RNF144A localizes primarily to cytoplasmic vesicles and plasma membrane where it interacts with cytoplasmic DNA-PKcs .

• Regulatory function: RNF144A functions by:

  • Binding to DNA-PKcs through amino acids 173-252

  • Promoting ubiquitination of DNA-PKcs both in vitro and in vivo

  • Targeting DNA-PKcs for degradation, which modulates the balance between DNA repair and apoptosis

• Functional outcome: By promoting DNA-PKcs degradation, RNF144A shifts cellular response toward apoptosis rather than DNA repair, suggesting a tumor suppressor function .

In zebrafish models, researchers can exploit the conservation of these pathways to investigate DNA damage responses in a vertebrate system with genetic and imaging advantages. RNF144A-B likely performs similar functions in zebrafish cells, though species-specific differences in regulation may exist.

What is known about RNF144A-B's role in neurodevelopment and brain disorders?

While direct evidence for RNF144A-B's specific role in zebrafish neurodevelopment is limited in the provided sources, several lines of reasoning support investigating this protein in neurological contexts:

• Zebrafish as neurological models: Zebrafish have emerged as valuable models for studying complex brain disorders due to their:

  • High physiological and genetic homology to humans

  • Similar CNS morphology

  • Ease of genetic manipulation

• Ubiquitination in neurological function: E3 ubiquitin ligases play crucial roles in:

  • Synaptic plasticity

  • Neuronal protein quality control

  • Neurodevelopmental pathways

• Research applications: Zebrafish models can be particularly valuable for investigating:

  • Autism spectrum disorders, which have high heritability (>90%)

  • Depression and stress responses, which involve altered protein degradation pathways

  • Neurodegenerative conditions where protein accumulation is pathogenic

Researchers interested in RNF144A-B's neurological functions should consider:

• Creating zebrafish knockdown/knockout models of rnf144ab

• Examining effects on brain development, neuronal morphology, and behavior

• Investigating interactions with neuronal proteins involved in stress responses or neurodegeneration

How can RNF144A-B be used in high-throughput screening approaches?

RNF144A-B can be effectively utilized in high-throughput screening (HTS) approaches to identify modulators of ubiquitination pathways:

• Assay development strategies:

  • FRET-based ubiquitination assays: Using fluorescently labeled ubiquitin and substrates to monitor transfer reactions in real-time

  • Cell-based reporter systems: Developing stable zebrafish cell lines expressing RNF144A-B and fluorescent substrates

  • Automated imaging platforms: Exploiting zebrafish transparency for in vivo visualization of protein dynamics

• Screening applications:

  • Identify small molecule inhibitors or enhancers of RNF144A-B activity

  • Discover novel substrates through proteomic approaches

  • Screen for genetic modifiers using CRISPR libraries

• Advantage of zebrafish platform:

  • Zebrafish larvae are amenable to HTS approaches for in vivo validation

  • Multiple behavioral tests can be automated for phenotypic screening

  • Combines vertebrate relevance with throughput capacity not possible in mammalian models

This approach is particularly valuable for identifying compounds that might modulate apoptotic responses during DNA damage, with potential applications in cancer research and neurodegenerative disease studies.

What are the key protein characteristics of recombinant RNF144A-B?

Below is a comprehensive data table summarizing the key characteristics of recombinant Danio rerio RNF144A-B protein:

Data compiled from product information

This comprehensive characterization provides researchers with essential parameters for experimental design and troubleshooting when working with recombinant RNF144A-B.

How do mutations in RNF144A-B affect its E3 ligase activity?

Mutational analysis provides critical insights into the structure-function relationship of RNF144A-B:

• Critical catalytic residues:

  • RING1 domain mutations (C20A/C23A): Completely abolish E3 ligase activity by disrupting the RING1 domain structure needed for E2 recruitment

  • RING2 domain mutation (C198A): Eliminates the RING/HECT-hybrid activity by removing the catalytic cysteine that accepts ubiquitin from E2 before transfer to substrate

• Functional consequences:

  • Wild-type RNF144A promotes ubiquitination of DNA-PKcs both in vitro and in vivo

  • Neither the RING1-dead mutant (C20A/C23A) nor the RING2-inactive mutant (C198A) can promote ubiquitination of DNA-PKcs

  • These mutations specifically affect catalytic activity without necessarily disrupting substrate binding

• Experimental applications:

  • These catalytically inactive mutants serve as essential negative controls in ubiquitination experiments

  • They allow researchers to distinguish between ubiquitination dependent on RNF144A-B activity versus non-specific or E2-dependent ubiquitination

  • The distinct mutations can help elucidate the mechanistic details of the RING/HECT-hybrid ubiquitination mechanism

These findings demonstrate that both RING domains are essential for RNF144A-B function, consistent with the current understanding of RBR E3 ligases as functioning through a RING/HECT hybrid mechanism rather than a simple RING-based mechanism.

How can researchers troubleshoot inconsistent results in RNF144A-B experiments?

When encountering inconsistent results in RNF144A-B experiments, researchers should systematically troubleshoot using the following methodological approach:

• Protein quality issues:

  • Verify protein integrity by SDS-PAGE analysis

  • Check for degradation using Western blotting with anti-His antibody

  • Monitor aggregation using size exclusion chromatography

  • Ensure proper folding through circular dichroism or limited proteolysis

  • Avoid repeated freeze-thaw cycles that may compromise activity

• Experimental conditions optimization:

  • Buffer components: Test different pH values (7.5-8.5) and salt concentrations

  • Cofactors: Ensure proper concentrations of DTT/ZnCl₂ for maintaining RING domain structure

  • Temperature: Compare activity at different temperatures (25°C vs. 37°C)

  • Time course: Extend reaction times to capture slower ubiquitination kinetics

• E2 enzyme compatibility:

  • Test multiple E2 enzymes as RBR E3 ligases may have specific E2 preferences

  • Include UbcH5 family members which often work with RBR ligases

  • Verify E2 activity with control substrates

• Controls and validation:

  • Always include positive controls (known active E3 ligase)

  • Use catalytically inactive mutants (C20A/C23A or C198A) as negative controls

  • Confirm substrate integrity and binding capability

  • Validate results using orthogonal approaches (cell-based assays in addition to in vitro studies)

• Technical considerations:

  • Fresh preparation of ATP-containing reaction buffers

  • Pre-clearing components to remove non-specific binding

  • Optimizing antibody concentrations for detection

By systematically addressing these potential sources of variability, researchers can identify and resolve issues causing inconsistent results in RNF144A-B experiments.

How might zebrafish RNF144A-B studies inform human disease research?

Zebrafish RNF144A-B research has significant translational potential for human disease studies:

• Cancer biology applications:

  • RNF144A functions as a regulator of DNA-PKcs, promoting its degradation and shifting cellular response toward apoptosis rather than DNA repair

  • This pro-apoptotic function suggests a potential tumor suppressor role that can be studied in zebrafish cancer models

  • Research shows that RNF144A depletion attenuates adriamycin-induced apoptosis, and its restoration restores sensitivity

• Neurodevelopmental disorder insights:

  • Zebrafish are increasingly used to model complex brain disorders including autism and other neurodevelopmental conditions

  • The zebrafish genome contains homologous regions to human loci associated with autism spectrum disorders

  • E3 ubiquitin ligases play crucial roles in neuronal development and function, making RNF144A-B a candidate for investigation in these contexts

• Methodological advantages:

  • Zebrafish combine vertebrate relevance with experimental accessibility

  • Their rapid development and optical transparency facilitate real-time observation of biological processes

  • High genetic homology to humans (~70% of human genes have at least one zebrafish ortholog) enhances translational value

• Drug discovery applications:

  • Zebrafish are amenable to high-throughput screening approaches

  • Compounds that modulate RNF144A-B activity could be rapidly tested for effects on relevant phenotypes

  • Behavioral assays in zebrafish can provide functional readouts for neurological effects

These translational applications position zebrafish RNF144A-B research as a valuable bridge between basic molecular studies and potential clinical applications in cancer, neurological disorders, and other conditions involving ubiquitin-dependent regulation.

What are the limitations of using recombinant proteins versus genetic models in RNF144A-B research?

Understanding the comparative advantages and limitations of recombinant protein approaches versus genetic models is critical for RNF144A-B research:

Recombinant Protein Approaches:

Advantages:

• Precise biochemical characterization of catalytic activity

• Direct measurement of substrate specificity and kinetics

• Controlled experimental conditions for mechanistic studies

• Ability to study specific domains through truncation or mutation

Limitations:

• Lack of cellular context and regulatory mechanisms

• Potential issues with proper folding or post-translational modifications

• Absence of physiological binding partners and subcellular localization

• May not reflect in vivo concentration and conditions

Genetic Models:

Advantages:

• Physiological expression levels and regulation

• Preservation of all cellular interactions and localization

• Ability to observe developmental and tissue-specific effects

• Capacity to identify novel functions and interactions

Limitations:

• Potential compensatory mechanisms obscuring phenotypes

• Difficulty isolating direct versus indirect effects

• Challenges in distinguishing catalytic from scaffolding functions

• Possible embryonic lethality with complete knockout

Complementary Strategy:
The most robust research approach combines:

• Recombinant protein studies to establish biochemical mechanisms

• Cell-based assays to verify cellular functions

• Genetic models to confirm physiological relevance

• Rescue experiments with mutant variants to establish specificity

This integrated approach leverages the zebrafish model's strengths in combining molecular accessibility with vertebrate physiology, providing a comprehensive understanding of RNF144A-B functions.