Recombinant Danio rerio ATPase family AAA domain-containing protein 1-A (atad1a)

What is Danio rerio and why is it suitable for ATAD1a research?

Danio rerio, commonly known as zebrafish, is a tropical fish originating from South Asia that has become a prominent vertebrate model organism in biomedical research. Zebrafish share approximately 70% of their genes with humans, including genes related to genetic diseases, making them valuable for studying protein function and disease mechanisms .

For ATAD1a research specifically, zebrafish offer several advantages:

• Rapid reproduction cycle (breeding every 2-3 days with up to 300 embryos per cycle)

• External fertilization allowing easy manipulation of embryos

• Fast development (major organs form within 24 hours)

• Transparent embryos enabling visualization of developmental processes

• Genome similarity to humans, including conserved protein degradation pathways

What is the general function of ATAD1 proteins in cellular processes?

ATAD1 belongs to the AAA-ATPase family of proteins that utilize ATP to facilitate protein degradation and quality control. Based on current research, ATAD1 plays critical roles in:

• Promoting disassembly and turnover of intermediate filaments, particularly desmin filaments in muscle tissue

• Facilitating protein degradation through interactions with the ubiquitin-proteasome system (UPS)

• Acting cooperatively with other proteolytic enzymes like calpain-1 to process substrates

• Contributing to normal protein turnover in tissues, with implications for both atrophy and growth regulation

ATAD1 is notable among AAA-ATPases for its preference for phosphorylated substrates, suggesting specialized roles in regulated protein degradation pathways .

What techniques can be used to identify ATAD1a binding partners in zebrafish models?

Based on established methodologies for ATAD1 research, three complementary approaches can be employed to identify binding partners:

• Size-exclusion chromatography (SEC):

  • Cross-link muscle tissue with 1% PFA

  • Homogenize and quench with glycine (0.125 M)

  • Precipitate proteins with ammonium sulfate

  • Fractionate using gel filtration column

  • Analyze fractions by SDS-PAGE, immunoblotting, and mass spectrometry

• Immunoprecipitation with high-stringency washing:

  • Homogenize tissue and obtain 6,000g supernatant

  • Immunoprecipitate ATAD1a using specific antibodies

  • Wash extensively with high-salt buffer (500 mM NaCl) to remove nonspecific interactions

  • Identify bound proteins by mass spectrometry

• AMP-PNP-based protein trapping:

  • Utilize non-hydrolyzable ATP analog (AMP-PNP) to trap ATPase-substrate complexes

  • Isolate protein complexes from tissue at different time points

  • Identify proteins that utilize ATP and bind to substrates of interest

These approaches collectively enabled identification of 32 ATAD1-interacting proteins in mammalian studies, providing a methodological blueprint for zebrafish research .

How can researchers assess ATAD1a function in zebrafish muscle degradation?

To investigate ATAD1a's role in muscle protein degradation in zebrafish models, researchers can employ the following methodological approaches:

• In vivo phosphorylation assays:

  • Isolate muscle tissue at different developmental or experimental timepoints

  • Fractionate into soluble and insoluble components

  • Analyze phosphorylation status of potential substrates using phospho-specific antibodies

  • Compare phosphorylation patterns between normal and ATAD1a-depleted conditions

• In vitro degradation assays:

  • Isolate insoluble fractions containing filamentous proteins

  • Subject to controlled degradation with recombinant calpain-1

  • Compare degradation efficiency with and without added ATAD1a

  • Monitor substrate processing through time-course analysis by Western blotting

• Genetic manipulation approaches:

  • Utilize morpholinos or CRISPR-Cas9 to knockdown or knockout ATAD1a

  • Assess muscle integrity through histological analysis

  • Measure protein levels of potential substrates

  • Evaluate muscle function through behavioral assays appropriate for zebrafish

What are the key protein interaction partners of ATAD1 identified in research?

Research has identified several critical interaction partners of ATAD1 that form functional complexes. While these were identified in mammalian systems, they provide valuable targets for investigation in zebrafish models:

The key interaction partners include:

• UBXN4 (UBX domain-containing protein 4): Contains a ubiquitin regulatory X domain that promotes recruitment of ubiquitinated substrates to AAA-ATPase complexes

• PLAA (Phospholipase A-2-activating protein): Contains WD40 repeats that bind ubiquitinated proteins, serving as an adaptor for substrate recognition

• Calpain-1: A calcium-dependent protease that cooperates with ATAD1 to facilitate desmin filament depolymerization

• Various proteasome components: Including PSMD4 (Rpn10), PSMC4 (Rpt3), and PSMC3 (Rpt5), which accumulate with ATAD1 on desmin filaments during degradation

• Ubiquitin-related enzymes: Including UBA1, UBE2L3, UBE2N, and E3 ligases like HUWE1

The table below summarizes key ATAD1 interaction partners identified through multiple proteomics approaches:

Data adapted from proteomic analysis of ATAD1 complexes

How does ATAD1a complex formation influence substrate specificity?

The ATAD1 complex composition appears to dynamically change based on cellular conditions, suggesting regulated substrate targeting. Experimental evidence indicates:

• The ATAD1-PLAA-UBXN4 complex forms a functional unit that promotes desmin filament depolymerization and degradation in atrophying muscle tissue

• ATAD1 shows preferential activity toward phosphorylated substrates, a unique feature among AAA-ATPases, likely mediated by its interaction partners containing phospho-Serine/Threonine-binding domains

• Different methodological approaches (SEC, immunoprecipitation, binding assays) identified partially overlapping but not identical sets of interaction partners, suggesting dynamic complex formation depending on cellular context

• ATAD1 appears to be recruited to substrates like desmin filaments early during degradation processes, then relocates to the cytosol as the substrate becomes solubilized

For zebrafish research, these findings suggest investigating both spatial and temporal aspects of ATAD1a complex formation during development or tissue remodeling processes.

What experimental approaches can resolve contradictions in ATAD1a functional data?

When faced with contradictory findings regarding ATAD1a function in zebrafish, researchers should consider these methodological approaches:

• Cellular compartment-specific analysis:

  • ATAD1 appears to shuttle between filamentous structures and cytosol during substrate processing

  • Separate analysis of soluble versus insoluble fractions can clarify seemingly contradictory roles

  • Time-course experiments can reveal sequential actions that might appear contradictory in endpoint analysis

• Genetic complementation studies:

  • Generate ATAD1a zebrafish knockouts

  • Attempt rescue with wild-type versus mutant versions (ATP-binding mutants, interaction partner binding mutants)

  • Compare with human ATAD1 complementation to assess conserved functions

• Substrate-specific approaches:

  • ATAD1 shows preferential activity toward phosphorylated substrates

  • Contradictory results might stem from different phosphorylation states of substrates

  • Comparing effects on phosphomimetic versus phospho-deficient substrate mutants can resolve mechanistic contradictions

• Interaction partner manipulation:

  • Knockdown specific ATAD1a binding partners (UBXN4, PLAA) individually

  • This can distinguish direct ATAD1a effects from those mediated by specific complex formations

  • May reveal context-dependent functions explaining contradictory observations

How can researchers effectively investigate ATAD1a's role in zebrafish development and disease models?

Zebrafish provide unique opportunities for investigating ATAD1a in development and disease contexts through these methodological approaches:

• Developmental time-course analysis:

  • Leverage zebrafish's rapid external development and transparency

  • Track ATAD1a expression and localization throughout development using fluorescent reporter lines

  • Correlate with developmental milestones of organ systems, particularly heart and muscle development

• Tissue-specific manipulation:

  • Utilize the Gal4-UAS system for tissue-specific ATAD1a knockdown or overexpression

  • Focus on tissues where mammalian ATAD1 shows functional importance (muscle, heart, nervous system)

  • Combine with live imaging to track immediate consequences of manipulation

• Disease model integration:

  • The zebrafish heart regenerates naturally, providing a model for studying ATAD1a in injury response

  • Generate transgenic lines with human disease-associated ATAD1 variants

  • Compare phenotypes with zebrafish ATAD1a knockout or knockdown models

• High-throughput screening approaches:

  • Utilize zebrafish embryos for medium-throughput drug screening

  • Identify compounds that modify ATAD1a activity or bypass ATAD1a deficiency

  • Test in phenotypic assays relevant to muscle integrity or heart function

What are the most promising approaches for characterizing novel functions of ATAD1a in zebrafish?

Based on current knowledge gaps, these approaches hold particular promise:

• Systematic substrate identification:

  • Adapt proximity labeling techniques (BioID, APEX) for use in zebrafish

  • Express ATAD1a fusion constructs in specific tissues

  • Identify tissue-specific interaction networks and potential novel substrates

• Investigating developmental stage-specific roles:

  • Generate conditional ATAD1a knockout zebrafish using technologies like heat-shock inducible Cre

  • Ablate ATAD1a function at different developmental stages

  • Assess immediate and long-term consequences for tissue development and homeostasis

• Comparative analysis across species:

  • Compare zebrafish ATAD1a function with mammalian ATAD1

  • Investigate conserved versus divergent interaction partners

  • Identify evolutionarily conserved core functions versus species-specific adaptations

• Integration with other protein quality control systems:

  • Investigate crosstalk between ATAD1a and other degradation pathways (autophagy, other AAA-ATPases)

  • Determine how these systems compensate for each other

  • Identify unique versus redundant functions of ATAD1a in protein homeostasis

How can ATAD1a research in zebrafish inform therapeutic strategies for muscle wasting conditions?

Zebrafish ATAD1a research has promising translational potential for muscle wasting conditions:

• Therapeutic target validation:

  • ATAD1 suppression attenuates muscle atrophy in mammalian models

  • Zebrafish provide a platform to validate these findings in a vertebrate model with high throughput

  • Combination with other genetic manipulations can identify synergistic therapeutic targets

• Drug discovery pipeline:

  • Screen for compounds that modulate ATAD1a activity or complex formation

  • Validate hits in zebrafish muscle atrophy models

  • Identify compounds that can penetrate tissue barriers and reach target tissues effectively

• Mechanism-based treatment strategies:

  • Since ATAD1 shows preferential activity toward phosphorylated substrates, investigate kinase inhibitors

  • Test compounds that modulate ATAD1a interaction with UBXN4 or PLAA

  • Develop approaches that selectively inhibit pathological versus physiological ATAD1a functions

• Biomarker development:

  • Identify ATAD1a-dependent degradation products that could serve as biomarkers

  • Validate in zebrafish models before testing in higher organisms

  • Develop assays to monitor ATAD1a activity in vivo for therapeutic monitoring