Recombinant Bovine DnaJ homolog subfamily B member 14 (DNAJB14)

What is the molecular structure of DNAJB14?

DNAJB14 contains a highly conserved J-domain followed by a glycine/phenylalanine-rich region and a transmembrane segment. It is a type II transmembrane protein with its J-domain facing the cytoplasmic side of the ER membrane. The J-domain contains the essential HPD motif that is critical for interaction with Hsc70/Hsp70 chaperones. DNAJB14 shares approximately 50% sequence identity with DNAJB12, another ER-localized J-protein, but DNAJB14's sequence is more highly conserved across species, with 93% identity between mouse and human homologs .

How does DNAJB14 function as a co-chaperone?

DNAJB14 acts as a co-chaperone with HSPA8/Hsc70 to promote protein folding and trafficking, prevent aggregation of client proteins, and facilitate the degradation of misfolded proteins through ERAD . Through its J-domain, DNAJB14 recruits Hsc70 to the ER membrane, which increases the ATPase activity of Hsc70. This interaction is crucial for the function of DNAJB14 in quality control mechanisms that target misfolded proteins for proteasomal degradation. DNAJB14 specifically enhances the degradation of misfolded membrane proteins but not misfolded luminal proteins, suggesting a specific role in membrane protein quality control .

What experimental methods are used to determine DNAJB14 membrane topology?

Researchers typically employ multiple complementary approaches to determine DNAJB14's membrane topology:

• Immunofluorescence microscopy to confirm ER localization

• Protease protection assays using microsomes to identify which domains are accessible to proteases

• Tag insertion (such as HA-tagging) at different positions followed by protease digestion

• Co-immunoprecipitation to verify interactions with cytosolic chaperones

For protease protection assays, microsomes from DNAJB14-expressing cells are isolated and treated with proteinase K (typically at 10 μg/ml) in the presence or absence of detergent (1% Triton X-100). Protection of specific domains from proteinase K digestion in the absence of detergent indicates their luminal localization .

What is the tissue-specific expression pattern of DNAJB14?

DNAJB14 mRNA is expressed at relatively low levels in most tissues compared to DNAJB12, which is ubiquitously expressed. In mice, DNAJB14 shows highest expression in testis, with lower levels detected in brain and muscle . This restricted expression pattern contrasts with DNAJB12's ubiquitous distribution, suggesting DNAJB14 may have specialized functions in specific tissues. The tissue-specific expression can be analyzed through RT-PCR using specific primers for DNAJB14, with appropriate normalization to housekeeping genes like β-actin .

How is DNAJB14 expression regulated under cellular stress?

DNAJB14 shows limited induction in response to heat stress, unlike many other heat shock proteins. Studies in NIH3T3 cells incubated at 42°C demonstrated minimal changes in DNAJB14 mRNA levels . This differs from the stress response of related chaperones, suggesting that DNAJB14 may be involved in constitutive rather than stress-induced quality control mechanisms. This behavior distinguishes DNAJB14 from other heat shock proteins that are typically upregulated during stress conditions and suggests specialized functions beyond typical stress responses.

What approaches can be used to modulate DNAJB14 expression experimentally?

Researchers can modulate DNAJB14 expression using several strategies:

When designing knockdown experiments, careful primer design is essential to ensure specificity for DNAJB14 over DNAJB12. For overexpression, researchers can use mammalian expression vectors like pCAGGS with DNAJB14 cDNA cloned into restriction sites (e.g., EcoRI) .

How does DNAJB14 facilitate ERAD of misfolded membrane proteins?

DNAJB14 facilitates ERAD through a multi-step process:

• DNAJB14, anchored in the ER membrane with its J-domain facing the cytosol, recognizes misfolded membrane proteins

• Through its J-domain, DNAJB14 recruits and binds to cytosolic Hsc70

• This interaction stimulates the ATPase activity of Hsc70, enhancing its chaperone function

• The DNAJB14-Hsc70 complex facilitates the extraction of misfolded membrane proteins from the ER

• The extracted proteins are then polyubiquitinated and targeted to the proteasome for degradation

This process is specific to membrane proteins, as DNAJB14 overexpression accelerates the degradation of misfolded membrane proteins like CFTRΔF508 but not misfolded luminal proteins. The DNAJB14-dependent degradation is inhibited by proteasome inhibitors like MG132, confirming that DNAJB14 enhances degradation through the ubiquitin-proteasome system .

What distinguishes DNAJB14's substrate specificity from other ER-associated J-proteins?

DNAJB14 exhibits distinct substrate specificity compared to other ER-associated J-proteins:

DNAJB14's specificity for membrane proteins over luminal proteins is likely due to its topology, with the J-domain facing the cytosol, allowing it to interact specifically with cytosolic Hsc70 and participate in the extraction of membrane proteins . The exact molecular determinants that dictate which specific membrane proteins are recognized by DNAJB14 versus DNAJB12 remain an area of active investigation.

How do researchers experimentally assess DNAJB14's impact on protein quality control?

Researchers employ several experimental approaches to assess DNAJB14's role in protein quality control:

• Pulse-chase experiments to measure degradation rates of model misfolded proteins (e.g., CFTRΔF508) in cells with normal, overexpressed, or knocked-down DNAJB14

• Co-immunoprecipitation to detect interactions between DNAJB14, Hsc70, and client proteins

• Proteasome inhibitor studies (using MG132) to confirm the involvement of the ubiquitin-proteasome system

• Microscopy to visualize localization of DNAJB14 with client proteins

• In vitro reconstitution of ERAD components to study the biochemical requirements for DNAJB14-mediated degradation

What is the role of DNAJB14 in SV40 viral infection?

DNAJB14 plays a critical role in simian virus 40 (SV40) infection:

• DNAJB14 is required for efficient SV40 infection in both semipermissive human cells and permissive monkey cells

• Knockdown of DNAJB14 causes a 7-fold reduction in SV40 infection in CV1 monkey cells and approximately 50-fold reduction in human cells

• DNAJB14 functions at an early stage of infection, specifically at the step where disassembling viral capsids exit from the ER lumen prior to nuclear entry

• The requirement for DNAJB14 is specific, as knockdown of the related J-protein DNAJB11 does not inhibit SV40 infection

These findings suggest that DNAJB14 is part of a specialized cellular machinery that SV40 hijacks to facilitate its entry and trafficking within host cells.

What experimental approaches are used to study DNAJB14's involvement in viral infection?

Researchers employ several specialized techniques to investigate DNAJB14's role in viral infection:

• RNA interference (shRNA) to knock down DNAJB14 expression in susceptible cell lines

• Viral infection assays measuring infection efficiency (e.g., by flow cytometry detecting viral protein expression)

• Transfection bypassing experiments to determine at which stage of viral entry DNAJB14 is required

• Confocal microscopy to track viral particle localization in cells with normal or reduced DNAJB14 levels

For example, researchers have used flow cytometry to measure SV40 infection rates in control cells versus cells expressing shRNAs that repress DNAJB14, demonstrating significant reduction in infection efficiency with DNAJB14 knockdown .

How does the interaction between DNAJB14 and viral components differ from its normal cellular functions?

The interaction between DNAJB14 and viral components represents a specialized adaptation of its normal cellular functions:

This comparison suggests that viruses like SV40 have evolved to hijack the DNAJB14-mediated translocation machinery, exploiting its ability to extract proteins from membranes while avoiding the degradation fate that would normally follow for cellular substrates .

What are DJANGOS and how do they relate to DNAJB14 function?

DJANGOS (DNaj-ANchored Granules Juxtaposed to Nuclear OrganelleS) are elaborate membranous structures that form within cell nuclei when DNAJB14 or DNAJB12 is overexpressed. Research has revealed important properties of DJANGOS:

• DJANGOS formation by DNAJB14 requires the presence of DNAJB12, suggesting a functional interaction between these proteins

• Conversely, DNAJB12 can induce DJANGOS formation even when DNAJB14 is knocked down

• The formation of these nuclear structures may represent a cellular adaptation to excess J-proteins or indicate previously unrecognized nuclear functions of these proteins

These findings suggest a hierarchical relationship between DNAJB14 and DNAJB12, with DNAJB12 playing a more fundamental role in ER membrane organization and DNAJB14 potentially acting in a more specialized capacity that depends on DNAJB12.

How can researchers differentiate between DNAJB14 and DNAJB12 functions?

Differentiating between DNAJB14 and DNAJB12 functions requires careful experimental design:

• RNA interference with highly specific siRNAs or shRNAs targeting unique regions of each transcript

• CRISPR-Cas9 knockout of each gene individually, followed by rescue experiments

• Expression of chimeric proteins containing domains from both DNAJB14 and DNAJB12

• Tissue-specific analyses focusing on contexts where one protein is expressed but the other is minimal (e.g., testis for DNAJB14)

• Comparative analysis of stress responses, as DNAJB12 shows significant stress induction while DNAJB14 does not

For example, researchers have established cell lines with reduced DNAJB12 or DNAJB14 expression using shRNAs, then examined the ability of each protein to compensate for the other's function in processes like DJANGOS formation or viral infection .

What are the potential implications of DNAJB14 research for protein misfolding diseases?

Research on DNAJB14 has several important implications for protein misfolding diseases:

• DNAJB14's ability to enhance the degradation of CFTRΔF508, a mutant protein associated with cystic fibrosis, suggests potential therapeutic applications for enhancing clearance of disease-causing mutant membrane proteins

• Understanding the molecular mechanisms by which DNAJB14 recognizes misfolded membrane proteins could inform the development of targeted therapies for diseases involving membrane protein quality control defects

• The tissue-specific expression of DNAJB14, particularly in testis, may provide insights into tissue-specific manifestations of protein misfolding diseases

• Comparative studies of DNAJB14 and DNAJB12 may reveal redundancies in the protein quality control system that could be therapeutically exploited

Translational applications might include modulating DNAJB14 expression or activity to enhance the clearance of disease-associated misfolded membrane proteins or developing small molecules that mimic DNAJB14's substrate recognition capabilities.