Recombinant Xenopus laevis DnaJ homolog subfamily B member 6-A (dnajb6-a)

What is the basic structure of Xenopus laevis DNAJB6-A?

Xenopus laevis DNAJB6-A is a molecular chaperone protein comprising 250 amino acids in its recombinant form, with a sequence that includes several functional domains: the J-domain, the G/F-rich domain, the S/T-rich domain, and the C-terminal domain (CTD) . The protein's J-domain (colored green in structural analyses) contains the highly conserved HPD motif responsible for HSP70 interaction, while the G/F-rich domain (pink in visualizations) contains glycine-phenylalanine repeats important for substrate interactions . The amino acid sequence includes: MVEYYEVLGV QRNASADDIK KAYRRLALKW HPDKNPDNKD EAERRFKEVA EAYEVLSDSK KRDIYDKYGK EGLTNRGGGS HFDEAPFQFG FTFRSPDDVF RDFFGGRDPF SFDLFADDPF DDFFGRSRHR ANRSRPAGGG GGPFLSTFGG FPAFGPSFSP FDSGFSSSFG SFGGHGGHGG FTSFSSSSFG GSEMGNFRSV STSTKVVNGR RVTTKRIVEN GQERVEVEED GQLKSLTVNG KEQLLRLDNK .

How does DNAJB6 function in cellular systems?

DNAJB6 functions primarily as a co-chaperone of HSP70, exhibiting a stimulatory effect on HSP70's ATPase activity in both a dose-dependent and time-dependent manner . In cellular systems, it acts as an endogenous molecular chaperone for various proteins, particularly neuronal proteins including huntingtin . The protein plays critical roles in:

• Protein quality control mechanisms

• Suppressing aggregation of polyglutamine-containing, aggregation-prone proteins

• Reducing cellular toxicity and caspase-3 activity

• Organization of intermediate filaments (e.g., KRT8/KRT18 filaments in mammalian systems)

• Microtubule organization during mitosis, particularly at spindle poles

• Nuclear envelope-associated processes

What expression systems are optimal for recombinant Xenopus laevis DNAJB6-A production?

For research-grade recombinant Xenopus laevis DNAJB6-A, several expression systems have proven effective, with each offering distinct advantages:

The most commonly documented system is yeast expression, which produces His-tagged DNAJB6-A (aa 1-250) with >90% purity suitable for biochemical assays including ELISA . For applications requiring higher purity or specific modifications, researchers should consider downstream purification methods such as conventional chromatography techniques.

What methodologies are effective for studying DNAJB6-A interactions with HSP70?

To investigate the co-chaperone activity of DNAJB6-A with HSP70, researchers can employ several methodologies:

• ATPase Activity Assays: Measure the stimulatory effect of DNAJB6-A on HSP70's ATPase activity using colorimetric or radiometric assays. This demonstrates the dose-dependent and time-dependent manner of interaction .

• Co-immunoprecipitation (Co-IP): Using antibodies against either DNAJB6-A or HSP70 to precipitate protein complexes, followed by western blotting to detect interacting partners.

• Surface Plasmon Resonance (SPR): Quantitatively measure binding kinetics between DNAJB6-A and HSP70 under varying conditions.

• Fluorescence Resonance Energy Transfer (FRET): For real-time analysis of DNAJB6-A and HSP70 interactions in living cells.

• Mutational Analysis: Create point mutations in the critical HPD motif of the J-domain to validate specificity of interactions with HSP70 .

The stimulatory effect on HSP70 ATPase activity can be demonstrated through enzyme kinetics experiments where increasing concentrations of recombinant DNAJB6-A (10-500 nM) progressively enhance the rate of ATP hydrolysis by HSP70 (typically measured at 1 μM) .

How can Xenopus laevis DNAJB6-A be used to study mitotic spindle organization?

Xenopus laevis DNAJB6-A has been established as a critical factor in mitotic spindle organization, making it valuable for studying this process. Researchers can employ the following methodologies:

• Xenopus Egg Extract Systems: One of the most powerful approaches involves using CSF-arrested egg extracts. Depletion of endogenous xDNAJB6-L from these extracts results in spindle pole focusing defects, which can be rescued by adding back purified MBP-xDNAJB6-L . This experimental setup allows for direct manipulation of protein levels and real-time observation of spindle assembly.

• RanGTP Regulation Studies: GST-xDNAJB6-L-coated beads can be incubated in CSF-arrested egg extract with or without RanGTP, followed by western blot analysis to detect protein interactions (particularly with importin-β) . This approach reveals the RanGTP-dependent regulation of DNAJB6 during mitosis.

• Cell-Based Assays: In complementary approaches, researchers can perform RNAi-mediated silencing of DNAJB6 in cell cultures (e.g., HeLa cells expressing H2B-eGFP and α-tubulin-mRFP) to monitor mitotic progression through time-lapse fluorescence microscopy . Quantitative metrics include:

  • Time from nuclear envelope breakdown to anaphase onset (35.5% longer in DNAJB6-silenced cells)

  • Distribution of mitotic phases

  • Spindle pole width measurements

  • Spindle orientation relative to substrate

  • Centrosome attachment and alignment

What approaches can reveal DNAJB6-A's role in protein aggregation prevention?

DNAJB6 has demonstrated significant anti-aggregation activity, particularly against polyglutamine-containing proteins related to neurodegenerative diseases . Researchers can investigate this function using:

• In vitro Aggregation Assays: Purified recombinant DNAJB6-A can be added to aggregation-prone proteins (e.g., polyQ-expanded huntingtin fragments) to monitor aggregation kinetics through:

  • Thioflavin T fluorescence for amyloid formation

  • Dynamic light scattering for aggregate size distribution

  • Electron microscopy for aggregate morphology

  • Filter trap assays for SDS-insoluble aggregates

• Cell-Based Models: Establish cellular models expressing aggregation-prone proteins (such as polyQ-proteins) with or without co-expression of DNAJB6-A, and analyze:

  • Aggregate formation using fluorescence microscopy

  • Cell viability and apoptosis markers

  • Caspase-3 activity measurements

  • Proteostasis network activation

• Isoform-Specific Functions: Compare DNAJB6 isoforms (particularly isoforms A and B) in huntingtin aggregation inhibition assays, as evidence suggests isoform B specifically inhibits huntingtin aggregation .

• Multiplex Reverse Genetics Platform: A recently developed approach allows for simultaneous analysis of multiple protein misfolding models, enabling comprehensive assessment of DNAJB6-A's role across different aggregation-prone proteins and identification of common mechanisms .

How does DNAJB6-A interact with p150Glued and affect dynein/dynactin function?

DNAJB6 interacts with the dynactin subunit p150Glued in a RanGTP-dependent manner during mitosis. This interaction is crucial for proper spindle organization and can be studied through:

• Pulldown Assays: MBP-xDNAJB6-L-coated beads can be used in egg extracts with or without RanGTP to detect specific interaction with p150Glued. This approach has demonstrated that p150Glued is specifically pulled down by MBP-xDNAJB6-L only in the presence of RanGTP .

• Immunofluorescence Analysis: In cells with silenced DNAJB6, researchers can examine the localization of dynein/dynactin components (including p150Glued) at spindle poles and kinetochores to determine how DNAJB6 affects their recruitment and function.

• Functional Assays for Dynein Activity: Since the defects observed upon DNAJB6 silencing (spindle misorientation, centrosome detachment, spindle pole focusing defects, and spindle length increase) are compatible with altered dynein activity, researchers can perform direct assays of dynein function with and without DNAJB6-A .

• Domain Mapping: Truncation mutants of DNAJB6-A can help identify which domains are essential for p150Glued interaction, potentially revealing whether this function is independent of or related to the HSP70 co-chaperone activity.

How can Xenopus laevis DNAJB6-A be utilized in neurodegenerative disease research models?

DNAJB6 has been identified as "a Key Factor in Neuronal Sensitivity to Amyloidogenesis," making it valuable for neurodegenerative disease research . Researchers can approach this area through:

• Comparative Studies: Compare the anti-aggregation effects of Xenopus DNAJB6-A with human DNAJB6 on disease-associated proteins to identify conserved mechanisms and potentially species-specific differences in chaperone function.

• Animal Models: Develop transgenic Xenopus models with altered DNAJB6-A expression to study neurodevelopmental and neurodegenerative phenotypes in vivo.

• Multiplex Platforms: Use recently developed multiplex reverse genetics platforms to study DNAJB6-A's effects on multiple aggregation-prone proteins simultaneously, identifying common protective mechanisms .

• Cellular Models of Neurodegeneration: Establish neuronal cell models expressing disease-associated proteins (e.g., polyQ-expanded huntingtin, α-synuclein, TDP-43) with manipulated levels of DNAJB6-A to assess protective effects on:

  • Protein aggregation

  • Cellular toxicity

  • Caspase activation

  • Proteasome function

  • Autophagic flux

What is the significance of DNAJB6-A's nuclear envelope localization?

Recent research has revealed that DNAJB6 accumulates in foci at the nuclear envelope, suggesting potential roles in nuclear structure and function :

• Localization Studies: Researchers can employ high-resolution imaging techniques (super-resolution microscopy, electron microscopy) to precisely determine DNAJB6-A's localization relative to nuclear pore complexes and other nuclear envelope components.

• Nuclear Transport Assays: Given the interaction of DNAJB6 with the Ran pathway , researchers can investigate whether DNAJB6-A affects nuclear-cytoplasmic transport of proteins or RNA.

• Nuclear Envelope Integrity: Assess whether DNAJB6-A plays a role in maintaining nuclear envelope integrity during interphase or nuclear envelope breakdown and reassembly during mitosis.

• Protein Quality Control at the Nuclear Envelope: Investigate whether DNAJB6-A's localization at the nuclear envelope indicates a specialized role in quality control for nuclear envelope proteins, potentially in cooperation with the HSP70 system.

What are common challenges in expressing and purifying functional Xenopus laevis DNAJB6-A?

Researchers working with recombinant Xenopus laevis DNAJB6-A may encounter several technical challenges:

• Solubility Issues: The multiple domains of DNAJB6-A, particularly the disordered regions, can lead to solubility problems. Optimize expression conditions (temperature, induction time) and consider fusion tags (His, MBP, GST) to improve solubility .

• Protein Stability: DNAJB6-A may exhibit limited stability in solution. Include appropriate stabilizing agents (glycerol, reduced DTT) in buffers and store at -80°C in single-use aliquots.

• Functional Assessment: Confirm that the recombinant protein retains functional activity through HSP70 ATPase stimulation assays or protein aggregation prevention assays before using in complex experiments.

• Isoform Specificity: Be aware that expression constructs should clearly specify which isoform of DNAJB6-A is being produced, as functional differences exist between isoforms .

• Purification Approach: Conventional chromatography techniques are typically employed for purification, with affinity chromatography using the His-tag being the initial capture step, followed by ion exchange and/or size exclusion chromatography for higher purity .

How can researchers differentiate between HSP70-dependent and HSP70-independent functions of DNAJB6-A?

DNAJB6 exhibits both HSP70-dependent and potentially HSP70-independent functions, which can be distinguished through:

What are emerging areas of research for Xenopus laevis DNAJB6-A?

Recent findings suggest several promising directions for future research:

• Nuclear Pore Complex Biogenesis: Investigating DNAJB6-A's role in the surveillance of FG-Nups and interphase nuclear pore complex biogenesis represents an emerging frontier .

• RanGTP-Regulated Functions: Further exploration of how RanGTP regulates DNAJB6 during different cell cycle phases could reveal novel mechanisms of spatial protein regulation .

• Comparative Chaperone Networks: Comparing the chaperone networks in Xenopus with those in mammalian systems to identify conserved and divergent mechanisms of proteostasis.

• Therapeutic Applications: Exploring whether DNAJB6-A's anti-aggregation properties could be harnessed for therapeutic approaches to protein misfolding diseases .

• Development of Research Tools: Creating new tools based on DNAJB6-A's properties, such as sensors for protein misfolding stress or regulatable chaperone systems for experimental control of protein aggregation.