Recombinant Mouse DnaJ homolog subfamily B member 12 (Dnajb12)

How does mouse Dnajb12 compare to human DNAJB12?

Mouse Dnajb12 shares approximately 87-93% sequence identity with human DNAJB12, particularly in functional domains . The highest conservation is observed in:

• The J-domain region (nearly identical)

• The transmembrane domain

• Portions of the DUF1977 domain, especially the N-terminal region

• The C-terminal portion of the DUF1977 domain shows greater divergence

• Some variations exist in post-translational modification sites

• The mouse protein contains conserved cysteine residues at positions 329 and 363 that are functionally important

This high degree of conservation suggests that findings from mouse models are likely applicable to human systems, making recombinant mouse Dnajb12 valuable for comparative studies.

What expression systems are commonly used to produce recombinant mouse Dnajb12?

Several expression systems have been validated for producing recombinant mouse Dnajb12:

What is the role of Dnajb12 in ER stress responses and how can researchers study this function?

Dnajb12 plays a dual role in ER stress responses:

• Under mild stress conditions: Facilitates protein folding and prevents aggregation by recruiting Hsc70 to the ER surface, coordinating ER-associated and cytosolic chaperone systems

• Under severe ER stress: Becomes destabilized and is degraded by the proteasome, particularly during reductive stress induced by DTT

Research has demonstrated that Dnajb12 is uniquely sensitive to ER stress compared to other ER-associated chaperones. Time-course experiments with thapsigargin (Tg, 6 μM) showed reduction in Dnajb12 levels between 2-4 hours with near-complete depletion occurring between 8-24 hours .

Methodological approaches to study this function:

• Stress induction protocols: Compare effects of different stressors (DTT at 0.6mM, thapsigargin at 3-6μM, and tunicamycin). Studies show DTT rapidly destabilizes Dnajb12 (reducing half-life from 6h to ~1.5h), while tunicamycin has minimal effect

• Pulse-chase experiments: To track protein degradation kinetics during stress (as demonstrated in Fig. 1D of search result )

• Co-immunoprecipitation: To detect stress-induced interactions with BiP/Grp78 (4-fold increase in BiP association with FLAG-Dnajb12 was observed after DTT treatment)

• Domain analysis: Studies show the ER luminal DUF1977 domain, particularly Cys-363, is crucial for stress sensitivity

What experimental approaches are effective for studying Dnajb12's interaction with ERAD machinery?

Dnajb12 interacts with specific components of the ERAD machinery, particularly during stress conditions. Research shows that destabilized Dnajb12 is degraded by ERAD complexes containing HERP, Sel1L, and gp78, but not by those containing RMA1, CHIP, or HRD1 .

Recommended experimental approaches:

• siRNA-mediated depletion: Knockdown of specific ERAD components (HERP, Sel1L, gp78) followed by stress induction and monitoring of Dnajb12 stability. This approach demonstrated that depletion of gp78, Sel1L, and HERP suppressed DTT-induced Dnajb12 degradation

• Co-immunoprecipitation assays: Using tagged versions of ERAD components as bait:

  • When HERP-FLAG was used as bait, both Sel1L and Dnajb12 were detected

  • When MYC-Sel1L was used as bait, Dnajb12 was detected

  • Depletion of endogenous HERP reduced Dnajb12 association with Sel1L by ~60%

• Proteasome inhibitors: To confirm involvement of the ubiquitin-proteasome system in Dnajb12 degradation

• Domain mutation studies: Creating specific mutations in the DUF1977 domain (such as C363A) to analyze effects on ERAD interaction and stress sensitivity

How does Dnajb12 regulate apoptosis through BOK, and what techniques can be used to investigate this pathway?

Dnajb12 plays a critical role in regulating apoptosis through control of the BCL-2 family member BOK. Studies have revealed:

• Dnajb12 is required to maintain BOK at low levels in human hepatoma (Huh-7) liver cancer cells

• BOK was detected in complexes with Dnajb12 and gp78

• Depletion of Dnajb12 (either during reductive stress or by shRNA) leads to BOK accumulation and activation of Caspase 3, 7, and 9

• Loss of Dnajb12 sensitizes cells to death from proteotoxic agents and proapoptotic chemotherapeutics like LCL-161

Experimental approaches to investigate this pathway:

• Knockdown studies: Using siRNA or shRNA against Dnajb12 followed by analysis of:

  • BOK protein levels by Western blot

  • Caspase activation using specific antibodies or activity assays

  • Cell viability under normal and stress conditions

• Co-immunoprecipitation: To detect physical interactions between Dnajb12, BOK, and gp78

• Apoptosis assays: Following Dnajb12 depletion, researchers should measure:

  • Annexin V/PI staining by flow cytometry

  • PARP cleavage by Western blot

  • Mitochondrial membrane potential changes

• Rescue experiments: Re-expression of wild-type Dnajb12 in knockdown cells should restore normal BOK levels and cell survival, while expression of the stress-resistant C363A mutant may show different effects

What are the challenges and solutions in producing functional recombinant mouse Dnajb12 protein?

Producing fully functional recombinant mouse Dnajb12 presents several challenges due to its transmembrane domain and complex structure:

Challenges and Solutions:

• Transmembrane domain: The transmembrane domain (near amino acid 250) makes full-length protein expression difficult in bacterial systems.

  • Solution: Most commercial recombinant proteins use truncated versions (amino acids 1-210) that exclude the transmembrane domain but retain the J-domain

• Proper folding: The DUF1977 domain contains critical cysteine residues that affect stability.

  • Solution: Expression in eukaryotic systems (yeast, insect, or mammalian cells) promotes proper folding and disulfide bond formation

• Solubility issues: Full-length protein tends to aggregate.

  • Solution: Use of fusion tags (His, GST) and optimized solubilization buffers with mild detergents

• Authentication challenges: Confirming functionality of the recombinant protein.

  • Solution: Activity assays measuring ATPase stimulation of Hsc70 partner protein

For researchers needing to produce their own recombinant Dnajb12, the optimal approach depends on the experimental application:

How can researchers effectively study the differential roles of Dnajb12 compared to related Hsp40 family members?

Distinguishing the specific functions of Dnajb12 from related family members (particularly DNAJB14) requires careful experimental design:

Key research findings and methodological approaches:

• Functional specificity: Despite similarities, Dnajb12 and DNAJB14 have distinct functions:

  • Both facilitate potassium channel maturation, but at different steps in the process

  • DNAJB12 requirements in ER quality control-autophagy are not shared by DNAJB14

  • DNAJB12 is degraded during ER stress while DNAJB14 remains stable

• Domain analysis approaches:

  • Sequence alignment of the DUF1977 domains of Dnajb12 and DNAJB14 reveals high identity in N-terminal regions but divergence in C-terminal portions

  • Creation of chimeric proteins by swapping domains between family members can identify regions responsible for functional differences

• Differential stress response study:

  • Challenge cells with DTT (0.6mM) or thapsigargin (3-6μM) and compare stability of Dnajb12 versus DNAJB14

  • The C-terminal portion of Dnajb12's DUF1977 appears responsible for stress sensitivity not shared by DNAJB14

• Specific knockdown experiments:

  • Selective siRNA targeting each family member followed by analysis of:

    • Effects on specific client proteins (e.g., potassium channels)

    • Impacts on ER stress responses

    • Changes in mitochondrial dynamics

Research has shown that when DNAJB12 was depleted in human cells, there was a "dramatic threefold increase in the quantity of nascent B-form CFTR that is converted to the C-form" , demonstrating its specific role in CFTR biogenesis that isn't compensated by other Hsp40 proteins.

What is the role of Dnajb12 in mitochondrial function and how can this be experimentally evaluated?

Recent research has revealed a previously unrecognized role for Dnajb12 in mitochondrial function:

• Cells with genetic knockout of DNAJB12 exhibit altered kinetics of phosphorylated Drp1 in response to stress caused by CCCP treatment

• DNAJB12 expression is regulated in response to mitochondrial potential uncoupler CCCP

• DNAJB12-depleted cells show increases in mitochondrial count and branching

Experimental approaches to investigate mitochondrial roles:

• Mitochondrial morphology analysis:

  • Confocal microscopy with MitoTracker dyes in wild-type versus Dnajb12 knockout/knockdown cells

  • Quantification of mitochondrial parameters (count, branching, volume)

• Mitochondrial dynamics protein analysis:

  • Western blot analysis of key mitochondrial dynamics proteins (Drp1, phospho-Drp1, Mfn1/2, OPA1) following Dnajb12 manipulation

  • Time-course studies with mitochondrial stress inducers (CCCP)

• Mitochondrial stress response:

  • Analysis of PINK1 stabilization patterns in wild-type versus Dnajb12-depleted cells during CCCP exposure

  • Measurement of mitochondrial membrane potential using fluorescent indicators

• Functional mitochondrial assays:

  • Oxygen consumption rate measurements

  • ATP production

  • Mitochondrial calcium handling

This emerging research suggests that Dnajb12, despite being ER-localized, plays important roles in ER-mitochondria communication and mitochondrial quality control.

What are the most effective antibody validation strategies for mouse Dnajb12?

Proper antibody validation is crucial for reliable Dnajb12 research. Based on available research methodologies:

Recommended validation strategies:

• Knockdown/knockout controls:

  • Use of siRNA, shRNA, or CRISPR-mediated knockout of Dnajb12 as negative controls

  • This approach has been successfully employed in several studies

• Recombinant protein controls:

  • Use of purified recombinant mouse Dnajb12 as a positive control

  • Competition assays with recombinant protein fragments at 100x molar excess for blocking experiments

• Applications-specific validation:

  • For Western blotting: Verify band at the expected molecular weight (40-42 kDa)

  • For immunoprecipitation: Confirm specific enrichment compared to IgG controls

  • For immunohistochemistry: Include peptide blocking controls

• Cross-reactivity testing:

  • Test against related family members (especially DNAJB14)

  • Assess specificity across species when using in comparative studies

Validated antibody applications from literature:

For pre-incubation blocking experiments, researchers should use recombinant protein fragments at 100x molar excess based on antibody concentration and incubate the mixture for 30 minutes at room temperature .