DNAJB6 Human

What is DNAJB6 and what is its primary function in human cells?

DNAJB6 is a human J-domain protein (JDP) that functions as a cellular protein quality control factor. Its primary role involves preventing various peptides from forming disease-associated amyloid aggregates, which are highly ordered fibrous protein structures linked to numerous neurodegenerative conditions .

As a co-chaperone of HSP70, DNAJB6 operates within the HSP70 machinery, though it possesses unique properties that distinguish it from other J-domain proteins. DNAJB6 has demonstrated exceptional ability to prevent aggregation of amyloidogenic substrates at remarkably low concentrations (as little as 1:100 DNAJB6 to substrate ratio), suggesting it primarily targets early nucleation events in the aggregation pathway .

DNAJB6 is ubiquitously expressed across tissues under non-stress conditions and has been implicated in various cellular processes including cell cycle regulation, placental development, and neural stem cell maintenance .

What are the two main isoforms of DNAJB6 and how do they differ?

DNAJB6 exists in two major isoforms: DNAJB6a and DNAJB6b, which differ primarily in their subcellular localization and length:

• DNAJB6a: The longer isoform (326 amino acids) that contains a nuclear localization signal (NLS) at the far end of its C-terminal domain, making it predominantly nuclear .

• DNAJB6b: The shorter isoform (241 amino acids) that lacks the extended C-terminal region containing the NLS, resulting in both cytoplasmic and nuclear distribution .

Despite their different localizations, both isoforms share the same domain organization and anti-amyloid capabilities. Experimental evidence has shown that both isoforms can suppress amyloid formation, indicating that their biochemical effects are not confined to specific cellular compartments .

How is DNAJB6 expression regulated in human cells?

DNAJB6 expression regulation involves several mechanisms:

What cellular processes is DNAJB6 involved in?

DNAJB6 participates in numerous essential cellular processes:

• Protein Quality Control: DNAJB6 prevents aggregation of amyloidogenic proteins including polyglutamine (polyQ) proteins, Aβ42 peptide, α-synuclein, and dipeptide repeats associated with neurodegenerative diseases .

• Nuclear Pore Complex (NPC) Biogenesis: DNAJB6 is essential for proper NPC assembly, localizing to herniations at the nuclear envelope when NPC biogenesis is stalled. In its absence, membrane stacks containing partly assembled NPCs accumulate in the cytoplasm, and nucleocytoplasmic transport becomes impaired .

• Development and Differentiation: DNAJB6 is crucial during embryonic development, particularly in placental formation and neural tube closure. Its deletion leads to failure of chorioallantoic fusion necessary for normal placenta development .

• Cytoskeletal Regulation: DNAJB6 binds directly to keratin 18 (K18) intermediate filaments and is essential for their degradation during development. Compromised DNAJB6 function leads to disorganization of K18 filament structures and toxic keratin inclusion formation .

• Neural Stem Cell Maintenance: DNAJB6 is involved in neural stem cell renewal and proliferation. Its absence leads to smaller neural tubes with thinner neuroepithelium, reflecting reduced ability of neural stem cells to proliferate and maintain their progenitor identity .

How does DNAJB6 prevent amyloid formation?

DNAJB6 employs several mechanisms to prevent amyloid formation:

• Nucleation Inhibition: In vitro studies revealed DNAJB6 primarily affects primary nucleation of amyloid formation. It acts on early nucleation events during the initial steps of aggregation reactions, suppressing their transition into amyloids .

• Secondary Prevention Mechanisms: Beyond primary nucleation inhibition, DNAJB6 can also inhibit secondary nucleation from seed fibrils and prevent fibril elongation .

• Substoichiometric Activity: DNAJB6 demonstrates remarkable efficacy at very low concentrations (as little as 1:100 DNAJB6 to substrate ratio), suggesting it targets specific nucleation-prone species rather than monomers .

• S/T-domain Dependent Mechanism: DNAJB6's unique serine/threonine-rich (S/T) domain is crucial for its anti-amyloid activity. This domain enables DNAJB6 to form oligomers and block nucleation and assembly of amyloid by several disease-associated proteins .

• HSP70 Cooperation: While DNAJB6 can directly prevent amyloid formation, its full anti-aggregation potential requires cooperation with the HSP70 machinery, to which DNAJB6 connects through its J-domain .

What is the significance of the S/T-domain in DNAJB6's anti-amyloid activity?

The serine/threonine-rich (S/T) domain is a unique feature of DNAJB6 that plays a critical role in its anti-amyloid activity:

• Structural Basis: The S/T-domain is located in DNAJB6's C-terminal region and provides the protein with its distinctive ability to prevent amyloid formation by various substrates .

• Substrate Specificity: Research has shown the S/T-domain is essential for DNAJB6's interaction with amyloidogenic substrates. It enables DNAJB6 to directly act on polyQ sequences, unlike most chaperones which require polyQ-flanking sequences to counteract aggregation .

• Oligomerization Support: The S/T-domain contributes to DNAJB6's ability to form oligomers, which is important for its potent activity in blocking nucleation and assembly of amyloid by disease-associated proteins .

• Differential Requirements: Interestingly, while the S/T-domain is crucial for preventing aggregation of amyloidogenic substrates, it appears dispensable for preventing aggregation of non-amyloid aggregates. For example, when preventing aggregation of parkin C289G mutant (which forms non-amyloid like aggregates), DNAJB6 does not require its S/T-domain but instead fully relies on HSP70 interaction .

• Experimental Validation: The critical role of the S/T-domain has been validated through domain deletion and mutation studies, where removal or alteration of this domain significantly reduces DNAJB6's ability to prevent amyloid formation while leaving other functions intact .

How does DNAJB6 interact with HSP70 in the chaperone machinery?

DNAJB6's interaction with HSP70 involves several coordinated mechanisms:

• J-domain Mediated Binding: Like all J-domain proteins, DNAJB6 binds to and regulates HSP70 through its N-terminal J-domain . This interaction stimulates HSP70's ATPase activity, which is essential for the chaperone cycle.

• Co-chaperone Function: DNAJB6 acts as a co-chaperone of HSP70 and requires the entire HSP70 machinery for its full anti-aggregation potential .

• Autoinhibition Regulation: Evidence suggests that DNAJB6, like other JDPs, may have a helix 5 region that can compete with HSP70 for binding to the J-domain, potentially influencing autoinhibition strength by preventing HSP70 binding .

• Selective HSP70 Recruitment: For amyloidogenic substrates, DNAJB6 relies partially on its interaction with HSP70, while for more amorphous aggregates (like parkin C289G), it fully depends on HSP70 interaction .

• HSF1 Regulation Link: Under chronic stress conditions, elevated DNAJB6 levels appear to titrate more HSP70 to its chaperoned substrates, thereby releasing HSP70 from HSF1, which in turn becomes active to mount a stress response .

• Differential Dependence: Interestingly, while DNAJB6 requires HSP70 cooperation for its full anti-aggregation activity, its effectiveness against amyloidogenic substrates shows less dependence on HSP70 compared to its activity against non-amyloidogenic substrates .

What are the known substrates of DNAJB6 and how were they identified?

DNAJB6 interacts with diverse substrates that have been identified through various experimental approaches:

• Amyloidogenic Proteins:

  • Polyglutamine (PolyQ) Proteins: Initially discovered as DNAJB6 substrates through cellular overexpression studies and later confirmed in multiple model organisms including Xenopus, Drosophila, and mice .

  • Aβ42 Peptide: Identified through in vitro aggregation assays showing DNAJB6's ability to prevent Alzheimer's-related Aβ42 amyloid formation .

  • α-Synuclein: Found to be a DNAJB6 substrate through aggregation suppression studies related to Parkinson's disease models .

  • Dipeptide Repeats: Generated by C9orf72, linked to ALS, identified as DNAJB6 substrates through aggregation prevention assays .

  • Prion Proteins: Various yeast prions were identified as DNAJB6 substrates by showing that DNAJB6 could prevent or cure prion formation .

• Cytoskeletal Components:

  • Keratin 18 (K18): Identified as one of the first described substrates of Mrj (mouse ortholog of DNAJB6) through co-immunoprecipitation and functional studies showing direct binding to K18 intermediate filaments .

• Nuclear Pore Complex Components:

  • FG-Nucleoporins (FG-Nups): Recently discovered as native substrates of DNAJB6 through studies of nuclear pore complex biogenesis. FG-Nups have large disordered regions with FG motifs that are prone to aggregate, and DNAJB6 was shown to delay their aggregation by controlling their behavior when they form condensates .

• Non-Amyloid Aggregation-Prone Proteins:

  • Parkin C289G Mutant: Identified as a DNAJB6 substrate through investigations into whether DNAJB6 could suppress aggregation of proteins forming amorphous (non-amyloid) aggregates. This mutant causes juvenile Parkinsonism and forms non-amyloid-like aggregates in cells .

These substrates were generally identified through a combination of:

• Aggregation prevention assays

• Co-immunoprecipitation studies

• Loss-of-function experiments

• In vitro aggregation kinetics

• Genetic interaction studies in model organisms

How does DNAJB6 contribute to nuclear pore complex biogenesis?

DNAJB6's essential role in nuclear pore complex (NPC) biogenesis represents a significant discovery about its physiological function:

• Nuclear Envelope Herniation Association: DNAJB6 was found to be a resident of herniations at the nuclear envelope that arise when NPC biogenesis is stalled, suggesting its involvement in resolving biogenesis issues .

• Prevention of Annulate Lamellae Accumulation: In the absence of DNAJB6, membrane stacks containing partly assembled NPCs (known as annulate lamellae) accumulate in the cytoplasm, indicating DNAJB6's role in proper NPC assembly .

• Nucleocytoplasmic Transport Support: DNAJB6 depletion leads to partially impaired nucleocytoplasmic transport, highlighting its importance for functional NPC formation .

• FG-Nucleoporin Chaperoning: DNAJB6 has FG-Nucleoporins (FG-Nups) as its native substrates. These nucleoporins have large disordered regions with FG motifs that are essential for their functional state in forming the barrier between cytoplasm and nucleus .

• Condensate Behavior Control: FG-regions of nucleoporins are prone to aggregate, and DNAJB6 delays their aggregation by controlling their behavior when they form condensates - liquid-like droplets that can mature into more solid aggregates if not properly regulated .

• Pioneering Discovery: This finding represents the first evidence that a molecular chaperone is key to NPC assembly, expanding our understanding of both chaperone function and nuclear pore biogenesis mechanisms .

This role highlights DNAJB6's specialized function in chaperoning intrinsically disordered proteins that require their unstructured regions for functionality, positioning it as a critical factor for maintaining nuclear-cytoplasmic communication.

What experimental models have been used to study DNAJB6 function?

Researchers have employed diverse experimental models to investigate DNAJB6 function:

• Cellular Models:

  • Human cell lines: Used to study DNAJB6's role in preventing aggregation of polyQ proteins and other amyloidogenic substrates .

  • Patient-derived cells: SCA3 patient cells were utilized to examine DNAJB6's effect on endogenously expressed full-length polyQ proteins .

  • Primary neurons: Both unstressed primary neurons and those expressing amyloidogenic proteins have been used to study DNAJB6's protective and potentially toxic effects .

• Animal Models:

  • Mouse (Mrj knockout): Studies of the mouse ortholog Mrj have revealed DNAJB6's importance in placental development and embryonic tissues .

  • Xenopus: Used to demonstrate DNAJB6's ability to prevent polyQ aggregation .

  • Drosophila: Employed to study DNAJB6's protective effects against polyQ aggregation in vivo .

• Yeast Models:

  • S. cerevisiae: Used to investigate DNAJB6's anti-prion activity and its ability to cure yeast of endogenous prions .

  • Sis1-deficient yeast: Strains with reduced function of Sis1 (a yeast protein related to DNAJB6) have been used to study how DNAJB6 protects from prion toxicity .

• In Vitro Biochemical Systems:

  • Purified protein aggregation assays: Used to study the kinetics of how DNAJB6 prevents amyloid formation at substoichiometric ratios and its effects on primary nucleation, secondary nucleation, and fibril elongation .

  • Reconstituted chaperone systems: Employed to study the cooperation between DNAJB6 and HSP70 .

• Structural Studies:

  • Domain deletion and mutation analyses: Used to investigate the functional importance of specific DNAJB6 domains, particularly the S/T-domain and G/F-domain .

These diverse experimental approaches have collectively contributed to our current understanding of DNAJB6's multifaceted roles in protein quality control, development, and disease prevention.

How do mutations in the G/F-domain of DNAJB6 lead to Limb Girdle Muscular Dystrophy?

Mutations in the G/F-domain of DNAJB6 cause an autosomal dominant form of Limb Girdle Muscular Dystrophy (LGMD1D), representing a significant area of DNAJB6 research:

• Structural Impact: The G/F-domain is a stretch following the J-domain in DNAJB6. Mutations in this region likely alter the protein's functional properties, including potential changes to the interaction surface of helix 5 and its affinity with regions on the J-domain .

• Dominant Negative Effect: The autosomal dominant inheritance pattern suggests these mutations produce a dominant negative effect, where the mutant protein interferes with the function of the wild-type protein, rather than simply losing function .

• Tissue Specificity: Despite DNAJB6's ubiquitous expression, mutations predominantly affect skeletal muscle, suggesting tissue-specific roles or sensitivities to DNAJB6 dysfunction. This specificity may relate to the unique proteostasis challenges in long-lived, mechanically stressed muscle fibers .

• Functional Consequences: While the exact mechanism remains incompletely understood, research suggests these mutations may:

  • Alter DNAJB6's interaction with HSP70

  • Disrupt client protein handling

  • Affect DNAJB6 oligomerization

  • Impair specific muscle-relevant substrates

• Experimental Models: Understanding of the pathomechanism has been advanced through:

  • Patient muscle biopsies showing characteristic pathological features

  • Cell models expressing mutant DNAJB6

  • Animal models recapitulating aspects of the disease

• Therapeutic Implications: The study of how G/F-domain mutations cause LGMD1D has implications for developing therapeutic strategies targeting protein quality control mechanisms in muscular dystrophies .

This research area highlights the critical importance of specific DNAJB6 domains in tissue-specific functions and illustrates how subtle alterations in chaperone activity can lead to selective tissue pathology.

What are the mechanistic differences in how DNAJB6 prevents amyloid versus amorphous aggregation?

DNAJB6 demonstrates differential mechanisms when preventing amyloid versus amorphous protein aggregation:

• Domain Dependence:

  • Amyloid Aggregation: DNAJB6's S/T-domain is crucial for preventing aggregation of amyloidogenic substrates like polyQ, Aβ42, and α-synuclein .

  • Amorphous Aggregation: The S/T-domain is dispensable for preventing aggregation of non-amyloid substrates like parkin C289G mutant. For these substrates, DNAJB6 fully relies on its interaction with HSP70 through the J-domain .

• HSP70 Dependence:

  • Amyloid Aggregation: DNAJB6 shows partial independence from HSP70 when preventing amyloid formation, able to function at least partially through direct substrate interactions .

  • Amorphous Aggregation: Prevention of amorphous aggregates is entirely HSP70-dependent, suggesting DNAJB6 primarily functions as a substrate recruiter for HSP70 in these contexts .

• Substrate Specificity:

  • Amyloid Aggregation: DNAJB6 demonstrates extraordinary specificity and efficiency for amyloidogenic substrates, functioning at substoichiometric ratios (as low as 1:100) .

  • Amorphous Aggregation: For non-amyloid aggregates, DNAJB6 shows less substrate specificity - other cytoplasmic JDPs can also effectively prevent these aggregates in an HSP70-dependent manner .

• Aggregation Phase Targeting:

  • Amyloid Aggregation: DNAJB6 primarily affects primary nucleation but can also inhibit secondary nucleation from seed fibrils and fibril elongation .

  • Amorphous Aggregation: The mechanism appears to involve more classical chaperone functions, likely focusing on early misfolding intermediates rather than specific nucleation events .

• Experimental Observations:

  • Studies with parkin C289G mutant revealed that all tested cytoplasmic JDPs could prevent its aggregation in an HSP70-dependent manner, suggesting general JDP activity is sufficient for amorphous aggregates .

  • In contrast, DNAJB6 shows exceptional and specific activity against amyloidogenic substrates compared to other JDPs .

These mechanistic differences highlight DNAJB6's versatility as a chaperone and suggest it evolved specialized functions to address the particular challenges posed by amyloidogenic proteins while maintaining conventional chaperone capabilities.

How does DNAJB6 selectively recognize and prevent aggregation of intrinsically disordered proteins?

DNAJB6's ability to recognize and prevent aggregation of intrinsically disordered proteins (IDPs) represents a novel and specialized chaperone function:

• Recognition Mechanism:

  • Sequence-Specific Interactions: Evidence suggests DNAJB6 may recognize specific amino acid sequences or motifs within IDPs, particularly in polyQ stretches and FG-repeat regions of nucleoporins .

  • Conformational Sensing: Rather than binding to fully formed monomers, DNAJB6 appears to recognize early oligomeric species or specific conformational states that precede aggregation .

• Nucleoporin Chaperoning:

  • DNAJB6 has been identified as a chaperone for FG-Nucleoporins (FG-Nups), which contain large intrinsically disordered regions with FG motifs necessary for their functional state in nuclear pore complexes .

  • It controls the behavior of these nucleoporins when they form condensates, preventing their transition into irreversible aggregates while allowing their normal function in forming the nuclear-cytoplasmic barrier .

• Biological Significance:

  • This represents an important evolutionary adaptation, as many proteins contain intrinsically disordered regions that require their unstructured state for proper functioning .

  • Prior to this discovery, it was unclear whether IDPs required specialized chaperones to maintain their functional states while preventing inappropriate aggregation .

• Experimental Evidence:

  • Studies show DNAJB6 delays aggregation of several nucleoporins by controlling their behavior when they form condensates - liquid-like droplets that can mature into more solid aggregates if not properly regulated .

  • This reveals DNAJB6 as a chaperone specifically for natively disordered proteins that are at risk of aggregation while preserving their functional disorder .

• Expanding Chaperone Paradigm:

  • This function expands our understanding of the chaperone network, extending its scope beyond guiding the folding of structured proteins to maintaining the functional disorder of IDPs .

  • It suggests specialized chaperoning mechanisms have evolved to address the unique challenges posed by IDPs in the cellular environment .

This specialized function positions DNAJB6 at the intersection of protein quality control and phase separation biology, highlighting its role in maintaining the delicate balance between functional disorder and pathological aggregation.

What are the current challenges in studying DNAJB6's role in neurodegenerative diseases?

Investigating DNAJB6's role in neurodegenerative diseases faces several significant challenges:

Addressing these challenges requires developing new experimental models, analytical techniques, and conceptual frameworks to advance our understanding of DNAJB6's role in neurodegenerative disease processes.

How might targeting DNAJB6 be exploited for therapeutic interventions in amyloid-related diseases?

The potential therapeutic exploitation of DNAJB6 for amyloid-related diseases presents several promising strategies and considerations:

• Expression Enhancement Approaches:

  • Gene Therapy: Delivering DNAJB6 genes to affected tissues could enhance local expression and provide protection against amyloid formation. This approach has shown promise in animal models of polyQ diseases .

  • Transcriptional Activation: Identifying compounds that upregulate endogenous DNAJB6 expression could provide a less invasive alternative to gene therapy. This might involve targeting HSF1 or other transcription factors that regulate DNAJB6 .

• Functional Mimetics Development:

  • S/T-Domain Peptides: The critical role of the S/T-domain in anti-amyloid activity suggests that peptides mimicking this domain could potentially inhibit amyloid formation .

  • Small Molecule Screening: Compounds that either mimic DNAJB6 activity or enhance its function could be identified through high-throughput screens focused on preventing amyloid nucleation .

• Combination Therapies:

  • Chaperone Network Targeting: Simultaneously enhancing DNAJB6 and other complementary chaperones might provide synergistic effects on proteostasis .

  • Anti-Amyloid Cocktails: Combining DNAJB6-based approaches with other anti-amyloid strategies could address the issue of differential sensitivity of amyloid conformations to DNAJB6 .

• Disease-Specific Considerations:

  • Early Intervention: DNAJB6's ability to prevent nucleation suggests it would be most effective as an early intervention before significant amyloid formation has occurred .

  • Substrate-Tailored Approaches: Given DNAJB6's differential effectiveness against different substrates, disease-specific modifications to the basic approach may be necessary .

• Delivery Challenges:

  • Blood-Brain Barrier Penetration: For neurodegenerative diseases, effective delivery across the blood-brain barrier represents a major challenge that might be addressed through advanced delivery systems or alternative routes .

  • Tissue Targeting: Developing methods for tissue-specific delivery or activation of DNAJB6 could minimize potential side effects in tissues where elevated DNAJB6 might be detrimental .

• Safety Considerations:

  • Developmental Roles: Given DNAJB6's importance in development, therapeutic interventions would need careful timing and targeting to avoid interference with essential functions .

  • Dose Optimization: Finding the optimal therapeutic window where DNAJB6 provides protection without causing toxicity in unstressed neurons will be crucial .

While these approaches hold promise, their development requires further fundamental research to fully understand DNAJB6's mechanisms of action and tissue-specific functions in both health and disease contexts.