Recombinant Human DnaJ homolog subfamily C member 16 (DNAJC16), partial

What is DNAJC16 and what is its classification within the heat shock protein family?

DNAJC16 is a member of the DnaJ/HSP40 subfamily C of molecular chaperones. It contains the characteristic J-domain that defines the DNAJ family. Traditionally, heat shock proteins were classified based on molecular weight, but modern classification focuses on structural domains and evolutionary relationships. DNAJC16 belongs to type III (C) subfamily of DNAJ proteins, which typically have the J-domain but may lack other domains found in type I and II DNAJ proteins . Unlike many constitutively expressed chaperones, DNAJC16 shows a developmental expression pattern, being predominantly expressed during embryogenesis but often repressed in neonatal or infant stages .

What is known about DNAJC16 expression patterns and developmental roles?

DNAJC16 demonstrates a distinct developmental expression pattern, being predominantly expressed during embryogenesis but repressed in neonates or infants . This expression profile suggests it plays specialized roles during development rather than functioning as a general housekeeping chaperone. Studies in non-human species (such as Penaeus vannamei) show expression across multiple tissues with higher levels in lymphoid organs, suggesting potential immune-related functions . The temporal regulation of DNAJC16 expression indicates it may be involved in specific developmental processes, possibly related to embryonic protein quality control or cellular differentiation mechanisms.

How does DNAJC16 differ structurally and functionally from other DNAJ family members?

DNAJC16 contains both the characteristic J-domain and a thioredoxin domain, based on evidence from studies of its ortholog in P. vannamei . This domain architecture distinguishes it from other DNAJ proteins. While all DNAJ proteins share the conserved J-domain, DNAJC16 differs from DNAJA subfamily members that contain G/F-rich regions and zinc finger domains, and from DNAJB subfamily members that have G/F-rich regions but lack zinc finger domains. The developmental expression pattern of DNAJC16 also contrasts with many other DNAJ family members that show constitutive expression across tissues and developmental stages . These differences suggest DNAJC16 has evolved specialized functions distinct from other family members.

What are the recommended expression systems for recombinant human DNAJC16?

For bacterial expression of recombinant human DNAJC16, modified E. coli strains like BL21(DE3) with enhanced capabilities for disulfide bond formation are recommended . When designing expression constructs, physiologically-regulated promoters like proU may yield better results than strong constitutive promoters . The evidence suggests using fusion tags such as GST to improve solubility, and inducing expression in late-exponential or early stationary phase . To enhance proper folding, consider applying osmotic shock using high concentrations of sucrose (0.3-0.5M) to trigger stress responses that facilitate proper protein folding . Additionally, maintaining lower temperatures (16-20°C) during induction can slow protein folding and improve yield of correctly folded protein.

How can researchers optimize solubility of recombinant DNAJC16?

To maximize solubility of recombinant DNAJC16, researchers should implement multiple strategies. First, utilize solubility-enhancing fusion tags such as GST, MBP, or SUMO, including a flexible linker between the tag and DNAJC16 . Optimize expression conditions by lowering induction temperature to 16-20°C, reducing inducer concentration, and applying osmotic shock with sucrose to trigger stress responses that improve folding . Buffer optimization is also critical - include stabilizing additives like 5-10% glycerol, optimize salt concentration (typically 150-300mM NaCl), and test different pH conditions in the physiological range . If these approaches yield insufficient soluble protein, consider a domain-based approach by expressing individual domains separately or identifying and removing hydrophobic regions that contribute to aggregation.

What purification strategies yield high-quality recombinant DNAJC16 for functional studies?

A multi-step purification strategy is recommended for obtaining high-purity recombinant DNAJC16. Begin with affinity chromatography using the fusion tag (His-tag, GST, etc.) for initial capture . For His-tagged proteins, use immobilized metal affinity chromatography (IMAC) with gradient elution to separate different binding populations. Follow with intermediate purification using ion exchange chromatography based on the theoretical pI of DNAJC16, or hydrophobic interaction chromatography if DNAJC16 has sufficient hydrophobic patches. Complete purification with a polishing step using size exclusion chromatography to remove aggregates and ensure homogeneity . Throughout the purification process, include stabilizing agents such as glycerol (5-10%) to prevent aggregation, and consider adding low concentrations of non-ionic detergents if stability issues arise.

What assays are most appropriate for measuring DNAJC16 chaperone activity?

For assessing DNAJC16 chaperone activity, researchers should employ multiple complementary approaches. A primary assay is the ATPase stimulation assay, which measures DNAJC16's ability to stimulate the ATPase activity of Hsp70 proteins using colorimetric phosphate detection. Protein refolding assays provide functional evidence of chaperone activity by monitoring the refolding of thermally or chemically denatured model substrates (such as luciferase or citrate synthase) in the presence and absence of DNAJC16 and Hsp70. Protein aggregation prevention can be assessed by monitoring light scattering of aggregation-prone proteins with and without DNAJC16. To identify potential client proteins, employ pull-down assays followed by mass spectrometry, or use surface plasmon resonance to measure binding affinities. Based on findings in other species, assessment of DNAJC16's impact on cellular stress responses and apoptosis pathways may also provide valuable functional insights .

How does DNAJC16 interact with other components of the cellular chaperone network?

Based on our understanding of DNAJ proteins, DNAJC16 likely interacts with specific Hsp70 family members through its J-domain, stimulating their ATPase activity to facilitate client processing. Researchers should investigate which specific Hsp70 isoforms partner with DNAJC16 using co-immunoprecipitation and in vitro binding assays. DNAJC16 may also interact with nucleotide exchange factors (NEFs) to modulate Hsp70 activity . The extended chaperone network interactions will determine client fate - whether proteins undergo folding, degradation, or transfer to other chaperone systems. A key research priority is determining DNAJC16's substrate specificity compared to other DNAJ proteins, as this will provide insights into its unique cellular functions . Subcellular localization studies will help understand which compartment-specific Hsp70 systems DNAJC16 interacts with, as different DNAJ proteins are specialized for different cellular compartments.

What approaches can identify potential DNAJC16 client proteins and interaction partners?

To comprehensively identify DNAJC16 client proteins and interaction partners, researchers should employ multiple complementary techniques. Affinity purification coupled with mass spectrometry (AP-MS) can identify proteins that co-purify with tagged DNAJC16. For capturing transient interactions typical of chaperone-substrate relationships, proximity labeling approaches like BioID or APEX provide advantages by labeling proteins in the vicinity of DNAJC16 in living cells. Crosslinking mass spectrometry can identify direct binding interfaces between DNAJC16 and its partners. Functional screens using DNAJC16 overexpression or knockout can identify proteins whose stability or folding depends on DNAJC16. In vitro binding assays with purified components can validate direct interactions and determine binding affinities. Given DNAJC16's developmental expression pattern, these approaches should be applied across different developmental stages to capture stage-specific interactions .

How can CRISPR-Cas9 be optimized for studying DNAJC16 function?

For optimal CRISPR-Cas9 studies of DNAJC16, design multiple sgRNAs targeting different exons, prioritizing conserved domains like the J-domain. Use computational tools that predict off-target effects and editing efficiency. Consider different editing strategies based on your research questions: complete knockout to determine essentiality and general function, domain-specific mutations to understand the role of different protein regions, or epitope tagging for protein localization and interaction studies. Validate editing by sequencing and protein expression analysis, and control for off-target effects using rescue experiments with CRISPR-resistant DNAJC16 variants. Design appropriate functional readouts to measure cellular stress responses, developmental phenotypes, or chaperone network compensation. For developmentally expressed genes like DNAJC16, inducible CRISPR systems may be particularly valuable to control the timing of gene disruption and avoid potential compensation mechanisms .

What are the challenges in studying protein-protein interactions involving DNAJC16?

Studying DNAJC16 interactions presents several technical challenges. First, chaperone-substrate interactions are typically weak and transient, requiring specialized techniques like chemical crosslinking or rapid isolation methods. Proximity labeling approaches (BioID, APEX) can capture transient interactions in living cells. Second, distinguishing direct from indirect interactions requires careful experimental design - co-immunoprecipitation may identify components of larger complexes, necessitating in vitro binding assays with purified components to confirm direct interactions. Third, DNAJC16 interactions may be condition-dependent, occurring only under specific conditions like stress or particular developmental stages . This requires designing experiments to capture interactions under relevant physiological states. Finally, obtaining structural information of chaperone-substrate complexes is challenging and may require hybrid approaches combining cryo-EM, crosslinking-MS, and computational modeling. Focusing on stable domains for initial structural characterization can provide a foundation for understanding more dynamic interactions.

How might DNAJC16's developmental expression pattern inform experimental design?

DNAJC16's developmental expression pattern - high during embryogenesis but repressed in neonatal or infant stages - should fundamentally shape experimental design . First, choose appropriate model systems that allow access to embryonic tissues or developmental processes. Embryonic stem cells, organoids, or developmental model organisms like zebrafish may be more suitable than adult-derived cell lines. Second, implement temporal analysis by profiling DNAJC16 function across developmental stages to identify critical windows of activity. Third, compare DNAJC16 with other developmentally regulated chaperones to identify common patterns or unique features. Fourth, design conditional knockout or knockdown systems that can disrupt DNAJC16 function at specific developmental stages to avoid compensatory mechanisms. Fifth, correlate DNAJC16 expression with developmental events such as differentiation, migration, or morphogenesis to generate hypotheses about its specific functions. Finally, investigate whether DNAJC16 clients include developmentally regulated proteins with stage-specific expression patterns similar to DNAJC16 itself.

How does human DNAJC16 compare to its orthologs in model organisms?

Comparative analysis of DNAJC16 across species reveals both conserved and divergent features. The core J-domain is likely highly conserved across species, reflecting its fundamental role in Hsp70 interaction . In contrast, C-terminal regions may show more variation, potentially reflecting species-specific functions. Based on studies in shrimp, DNAJC16 contains a thioredoxin domain in addition to its J-domain, but this domain organization should be verified across species . The developmental expression pattern of DNAJC16 (embryonic expression followed by repression) appears to be a conserved feature , suggesting evolutionary conservation of its developmental role. For research purposes, different model organisms offer complementary advantages: mouse models for developmental studies, zebrafish for in vivo developmental visualization, C. elegans or Drosophila for genetic manipulation, and yeast for basic functional studies in a simplified chaperone network.

What can we learn from studies of DNAJC16 in non-mammalian organisms?

Studies of DNAJC16 in non-mammalian organisms have provided valuable insights that can guide human DNAJC16 research. In Penaeus vannamei (shrimp), PvDnaJC16 contains both DnaJ and thioredoxin domains, suggesting a potential role in redox regulation in addition to its chaperone function . PvDnaJC16 is upregulated during viral infection and appears to enhance hemocyte apoptosis, which accelerates viral spreading . This finding suggests human DNAJC16 might similarly modulate apoptotic pathways and potentially play roles in immune regulation or viral pathogenesis. The tissue distribution pattern in non-mammalian species (highest in lymphoid organs in shrimp) provides clues about potential functions in specific tissues . These comparative insights can inform hypothesis generation for human DNAJC16 research, particularly regarding potential roles in development, immune function, and stress response pathways.

What experimental systems are most appropriate for different research questions about DNAJC16?

The choice of experimental system should align with specific research questions about DNAJC16. For structural studies, bacterial expression systems optimized for soluble protein production are most appropriate, with E. coli BL21(DE3) strains and physiologically-regulated promoters like proU showing promise . For basic biochemical characterization of chaperone activity, purified recombinant DNAJC16 in reconstituted systems with Hsp70 and model substrates provides controlled conditions. To study developmental roles, embryonic stem cells, induced pluripotent stem cells, or developmental model organisms like zebrafish or Xenopus are valuable. For investigating tissue-specific functions, primary cells from relevant tissues or organoid systems may be more physiologically relevant than immortalized cell lines. Genetic screens to identify DNAJC16 clients or genetic interactors are most efficiently performed in systems amenable to high-throughput manipulation like yeast or cultured mammalian cells. When studying DNAJC16 in disease contexts, patient-derived samples or disease-specific cell models should be considered.

What are the common pitfalls in recombinant DNAJC16 expression and how can they be addressed?

Common pitfalls in recombinant DNAJC16 expression include protein insolubility, improper folding, and low yield. To address insolubility, use solubility-enhancing fusion tags like MBP rather than His-tags alone, and optimize expression conditions by lowering temperature (16-20°C) and inducer concentration . Apply osmotic shock with sucrose (0.3-0.5M) to trigger stress responses that improve folding . For improperly folded protein, co-express with molecular chaperones or consider refolding protocols from inclusion bodies. If yields remain low, optimize codon usage for the expression host, use strains with rare tRNA supplements, and try different media formulations. Protein degradation can be addressed by including protease inhibitors throughout purification and testing different buffer conditions to enhance stability. When troubleshooting, analyze samples from each step using SDS-PAGE and activity assays to identify where losses or problems occur. Additionally, express truncated constructs or individual domains if the full-length protein proves particularly challenging.

How can researchers validate that recombinant DNAJC16 is properly folded and functional?

Validating proper folding and functionality of recombinant DNAJC16 requires multiple complementary approaches. First, assess biophysical properties through circular dichroism spectroscopy to evaluate secondary structure content, thermal shift assays to measure protein stability, and size exclusion chromatography to confirm proper oligomeric state. Second, verify biological activity through functional assays such as stimulation of Hsp70 ATPase activity and prevention of substrate protein aggregation. Third, confirm proper domain structure through limited proteolysis, which yields characteristic fragment patterns for well-folded proteins. Fourth, analyze binding to known interaction partners (particularly Hsp70) using techniques like surface plasmon resonance or isothermal titration calorimetry. Finally, compare the properties of recombinant DNAJC16 to native protein where possible, or to well-characterized related DNAJ proteins as benchmarks. Only protein that passes multiple quality control criteria should be used for downstream applications.

What quality control measures should be implemented when working with recombinant DNAJC16?

Rigorous quality control is essential when working with recombinant DNAJC16. Begin with identity verification through mass spectrometry to confirm the correct protein sequence and detect any post-translational modifications or truncations. Assess purity using multiple methods: SDS-PAGE with densitometry analysis (aim for >95% purity), size exclusion chromatography to detect aggregates or contaminating proteins, and dynamic light scattering to evaluate sample homogeneity. Confirm proper folding using circular dichroism spectroscopy, fluorescence spectroscopy, and thermal shift assays. Functional validation should include ATPase stimulation assays and client protein protection assays compared to established chaperone controls . Stability testing under different storage conditions is important - monitor activity over time and assess freeze-thaw stability. Implement batch-to-batch comparison to ensure reproducibility, and maintain detailed documentation of purification conditions and quality metrics for each preparation.

What are the latest findings regarding DNAJC16's potential roles in cellular stress and disease?

Recent studies suggest potential roles for DNAJC16 in cellular stress responses and disease pathways. Based on findings in Penaeus vannamei, DNAJC16 may modulate apoptotic responses during cellular stress, particularly during viral infection . The upregulation of PvDnaJC16 during viral infection enhanced hemocyte apoptosis, which accelerated viral spreading . This suggests human DNAJC16 might similarly influence cell fate decisions during stress conditions. Given DNAJC16's developmental expression pattern, it may have particular relevance to developmental disorders or early-onset diseases . Researchers should investigate whether DNAJC16 dysregulation contributes to neurodevelopmental disorders, as many chaperones play critical roles in neural development. Additionally, DNAJC16's potential roles in proteostasis suggest it might influence protein aggregation disorders, particularly those with developmental origins. Future studies should examine DNAJC16 expression and function in disease-relevant tissues and developmental contexts.

How might advances in structural biology enhance our understanding of DNAJC16?

Advances in structural biology promise to transform our understanding of DNAJC16 function. Cryo-electron microscopy (cryo-EM) can now resolve structures of challenging protein complexes, potentially capturing DNAJC16 in complex with Hsp70 and client proteins. AlphaFold and other AI-based structure prediction tools can generate increasingly accurate models of DNAJC16, particularly useful for regions lacking experimental structures. Integration of hydrogen-deuterium exchange mass spectrometry can map dynamic regions and conformational changes during DNAJC16's functional cycle. Time-resolved structural studies using techniques like time-resolved crystallography or cryo-EM could visualize the dynamics of DNAJC16-mediated protein folding. Crosslinking mass spectrometry can identify binding interfaces between DNAJC16 and its partners with residue-level precision. These approaches will help elucidate how DNAJC16's structure enables its developmental specificity and client selectivity, potentially revealing targetable sites for therapeutic intervention in relevant disease contexts.

What novel approaches could advance the study of DNAJC16's developmental roles?

Innovative approaches are needed to fully elucidate DNAJC16's developmental functions. Developmental single-cell RNA-seq and spatial transcriptomics can map DNAJC16 expression with unprecedented resolution, revealing specific cell populations and developmental stages where it functions . CRISPR-based lineage tracing combined with DNAJC16 knockout can determine how DNAJC16 affects cell fate decisions during development. Optogenetic control of DNAJC16 activity would allow precise temporal manipulation to identify critical windows of function. Client identification during specific developmental stages using stage-specific proximity labeling could reveal developmentally regulated substrates. Organoid systems derived from stem cells provide accessible models to study DNAJC16 in human development without the ethical constraints of embryo research. In vivo imaging of tagged DNAJC16 in transparent model organisms like zebrafish embryos could visualize its dynamics during development. These approaches would provide mechanistic insights into DNAJC16's developmental roles and potential connections to developmental disorders.

What resources are available to researchers studying DNAJC16?

Researchers studying DNAJC16 can access various specialized resources. For sequence and structure information, UniProt provides curated protein sequence and annotation, while the AlphaFold Database offers predicted structural models. Expression data is available through GTEx for tissue-specific expression patterns and the Human Protein Atlas for protein localization information. For interaction analysis, STRING provides predicted and known interaction networks, while BioGRID contains experimentally validated protein interactions. Functional analysis can be performed using DAVID for functional annotation, KEGG for pathway analysis, and Gene Ontology resources for functional categorization. Comparative genomics resources include Ensembl for genomic information and orthologs, and the UCSC Genome Browser for evolutionary conservation analysis. For experimental work, Addgene may have available DNAJC16 expression constructs, and commercial antibody resources should be validated carefully given the challenges of specific detection for chaperone family members.

How can researchers design effective detection methods for DNAJC16 in complex biological samples?

Designing effective DNAJC16 detection methods requires consideration of sensitivity, specificity, and sample complexity. For immunological detection, carefully validate antibodies against recombinant DNAJC16 and use knockout/knockdown controls to confirm specificity, as cross-reactivity with other DNAJ family members is a common issue. For mass spectrometry approaches, identify unique peptides that distinguish DNAJC16 from other family members, and develop targeted proteomics methods (PRM, MRM) for sensitive detection in complex samples. When quantifying DNAJC16 mRNA, design primers spanning exon-exon junctions for specificity and perform melt curve analysis to confirm single amplification products. For in situ detection in tissues, validate probe specificity using appropriate controls and optimize signal amplification for low-abundance detection. When studying developmental contexts, timing is critical - design sampling strategies that capture relevant developmental windows given DNAJC16's temporal expression pattern .

What bioinformatics approaches are most valuable for predicting DNAJC16 function and interactions?

Several bioinformatics approaches can provide insights into DNAJC16 function and interactions. Sequence-based analysis using multiple sequence alignment across species can identify conserved motifs and functional residues that have been maintained throughout evolution . Structure prediction using AlphaFold2 or RoseTTAFold can generate models of DNAJC16 domains to guide functional hypotheses. Domain analysis can identify functional modules and motifs that suggest specific activities or interactions. Molecular docking simulations can predict interactions with Hsp70 and potential clients. Network analysis using protein-protein interaction databases can place DNAJC16 within broader cellular pathways. Gene expression correlation analysis across developmental datasets can identify genes with similar expression patterns, suggesting functional relationships . Pathway enrichment analysis of predicted interactors can reveal biological processes DNAJC16 might regulate. Evolutionary rate analysis can identify functionally constrained regions under purifying selection. Together, these computational approaches can generate testable hypotheses about DNAJC16 function to guide experimental design.