DHX29 Antibody

What is DHX29 and what is its significance in cellular function?

DHX29 (DEAH-box helicase 29) is a 155 kDa protein belonging to the DExD/H-box helicase superfamily. It plays critical roles in both translation initiation and innate immunity. In human cells, the canonical protein consists of 1369 amino acid residues and is primarily localized in the cytoplasm .

DHX29 functions as:

• An essential translation initiation factor that helps ribosomes scan through highly structured 5′ UTRs

• A cytosolic nucleic acid cosensor that triggers RIG-I/MAVS-dependent antiviral signaling pathways

• A regulator of start codon selection during translation initiation

The protein is notably widely expressed across many tissue types but shows particularly specific expression in epithelial cells and fibroblasts compared to immune cell subsets .

What is the domain structure of DHX29 and how does it relate to function?

DHX29 consists of several distinct functional domains that contribute to its activity:

The unique architecture of DHX29 enables its specialized functions, with the NTR and winged-helix domains being particularly important for specific ribosomal targeting .

What are the optimal conditions for using DHX29 antibodies in Western blotting?

For optimal Western blot results with DHX29 antibodies:

• Sample preparation: Standard cell or tissue lysates prepared with RIPA buffer containing protease inhibitors are suitable.

• Gel conditions: Use 6-8% gels for better resolution of the 155 kDa DHX29 protein.

• Recommended dilutions: Most commercial DHX29 antibodies perform optimally at 1:1000 dilution for Western blotting .

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Incubation: Overnight at 4°C for primary antibody, followed by 1-2 hours at room temperature for secondary antibody.

• Detection: Both chemiluminescence and fluorescence detection methods are compatible.

When validating a new DHX29 antibody, compare expression levels between epithelial cells/fibroblasts (high expression) and immune cells like monocytes or T cells (low expression) as a specificity control .

How can DHX29 knockdown experiments be properly designed and validated?

When designing DHX29 knockdown experiments:

• siRNA selection: Use multiple individual sequence siRNAs targeting different regions of DHX29 mRNA. Research has shown that knockdown efficiency correlates directly with functional effects .

• Validation methods:

  • Western blotting to confirm protein reduction (target >75% knockdown)

  • qRT-PCR to measure mRNA levels

• Essential controls:

  • Non-targeting siRNA control

  • Positive control targeting a known essential gene

  • Rescue experiments with siRNA-resistant DHX29 constructs

• Expected phenotypes:

  • Decreased type I IFN and IL-6 production in response to cytosolic nucleic acids

  • Reduced production of IP-10 and IL-8

  • Impaired response to viral stimulation

  • Potential translation defects for mRNAs with structured 5′ UTRs

Research has demonstrated that knockdown efficiency >75% is necessary to observe significant functional effects on cytokine production .

What cell types are most appropriate for studying DHX29 function?

Based on expression patterns and functional studies, the following cell types are most appropriate for DHX29 research:

DHX29 shows dramatically specific expression in airway-derived epithelial cells and fibroblasts compared to immune cell subsets, making these cell types ideal for studying its natural function .

How does DHX29 contribute to innate immune responses against viral infections?

DHX29 plays a sophisticated role in innate immunity through several mechanisms:

• Nucleic acid sensing: DHX29 functions as a cytosolic nucleic acid cosensor that directly binds to both poly I:C (dsRNA mimic) and poly dA:dT (dsDNA mimic) .

• MAVS-dependent signaling: DHX29 interacts with RIG-I and MAVS through its helicase 1 domain to activate downstream signaling pathways, leading to type I IFN and inflammatory cytokine production .

• Virus-specific responses:

  • Acts as an RNA co-sensor specifically for MDA5-mediated EMCV detection

  • Enhances MDA5-dsRNA binding affinity

  • Preferentially interacts with structured RNAs

• Cell-type specificity: DHX29-dependent antiviral responses are particularly important in epithelial cells and fibroblasts, which are often the first cells to encounter viruses during infection .

Experimental evidence shows that DHX29 knockdown significantly reduces IFN-β and IL-6 production upon cytosolic nucleic acid and virus stimulation, highlighting its critical role in antiviral defense mechanisms .

What is the functional relationship between DHX29 and translation initiation?

DHX29 plays several critical roles in translation initiation:

• Scanning through structured 5′ UTRs:

  • Binds to 40S ribosomal subunits

  • Possesses 40S-stimulated NTPase activity

  • Helps ribosomal complexes navigate through stable RNA secondary structures that cannot be resolved by eIF4A/4B/4G alone

• Start codon selection:

  • Stimulates recognition of AUG start codons but not near-cognate CUG codons

  • This effect is independent of the nucleotide context

  • Depends on contact between DHX29 and eIF1A

• Ribosomal accommodation:

  • Prevents stems from incorrectly entering the mRNA-binding channel

  • Promotes proper threading of mRNA through the exit portion

  • Suppresses formation of aberrant 48S complexes

• Structural requirements:

  • NTP hydrolysis is essential for DHX29's function in initiation

  • The OB domain plays a critical role in regulating NTPase activity

Recent research has revealed that DHX29 may directly influence viral mRNA translation, such as in HCMV infection where it regulates the expression of immediate early proteins IE1 and IE2 .

How can researchers distinguish between DHX29's role in translation versus innate immunity?

Distinguishing between DHX29's dual functions requires careful experimental design:

• Domain-specific mutations:

  • Mutations in the RecA1/RecA2 domains affect NTPase activity and translation function

  • The β-hairpin in RecA2 is particularly important for translation

  • The large RecA2 insert plays an autoinhibitory role in NTPase activity

• Selective pathway inhibition:

  • Use MAVS knockout or knockdown to eliminate innate immune signaling while preserving translation function

  • Employ translation-specific inhibitors in conjunction with DHX29 manipulation

• Temporal separation:

  • Translation effects are immediate

  • Innate immune signaling requires transcriptional responses (hours)

  • Monitor both immediate translational readouts and delayed immune markers

• Specific readouts:

  • Translation: polysome profiling, ribosome footprinting, reporter assays with structured 5′ UTRs

  • Immunity: type I IFN production, NF-κB activation, IRF3 phosphorylation

• Cell type selection:

  • Use cell types deficient in innate immune sensors but competent for translation to isolate translation effects

Recent studies have employed mutational analysis to successfully separate these functions, demonstrating that specific domains can be targeted to disrupt one function while preserving the other .

What factors might affect DHX29 antibody detection sensitivity and specificity?

Several factors can influence the reliability of DHX29 antibody-based detection:

• Antibody selection issues:

  • Epitope accessibility: DHX29's complex domain structure may result in epitopes being masked during certain interactions

  • Cross-reactivity: DHX29 belongs to the DExD/H-box helicase family with 59 members, increasing the risk of cross-reactivity

  • Lot-to-lot variability: Different production lots may show varying sensitivity

• Experimental conditions affecting detection:

  • Sample preparation: Harsh lysis conditions may disrupt epitopes

  • Cell type variation: DHX29 is constitutively expressed in epithelial cells but absent in many immune cell types

  • Post-translational modifications: May affect epitope recognition

• Optimization strategies:

  • Validate with positive controls (epithelial cells) and negative controls (immune cells)

  • Compare multiple antibodies targeting different epitopes

  • Use siRNA knockdown samples as specificity controls

  • Consider native versus denaturing conditions based on the antibody's properties

The specific reactivity pattern (human, mouse, rat) should be verified for each antibody, as most commercial DHX29 antibodies show cross-reactivity with these species .

How can researchers accurately interpret DHX29 functional assays in the context of viral infections?

Interpreting DHX29 functional assays in viral infection studies requires careful consideration:

• Distinguishing direct vs. indirect effects:

  • DHX29 affects both translation and innate immunity

  • Viral replication defects could result from either pathway

• Controls and comparisons:

  • Include RIG-I knockdowns as comparators for innate immune effects

  • Use translation factor knockdowns (non-immune related) to contextualize translation effects

  • Employ viral mutants that specifically target either translation or immune evasion

• Temporal analysis:

  • Early timepoints (0-6h): Focus on translation effects

  • Later timepoints (6-24h): Consider combined translation and immune effects

• Pathway-specific readouts:

  • Translation: viral protein synthesis rates, polysome association of viral mRNAs

  • Immunity: IRF3 phosphorylation, ISRE reporter activation, IFN production

• Virus selection considerations:

  • RNA viruses (like EMCV): DHX29 may function as a co-sensor with MDA5

  • DNA viruses (like HCMV): DHX29 may primarily affect translation of structured viral transcripts

Recent research has demonstrated that DHX29 depletion can reduce HCMV replication by decreasing the translation efficiency of immediate early protein mRNAs, highlighting the importance of examining translation-specific endpoints .

What are the key considerations when using DHX29 antibodies for studying protein-protein interactions?

When investigating DHX29 protein interactions:

• Antibody selection for interaction studies:

  • Choose antibodies targeting regions not involved in the predicted interaction

  • Validate that the antibody doesn't disrupt the interaction of interest

  • Consider epitope-tagged DHX29 constructs as alternatives

• Experimental approaches:

  • Co-immunoprecipitation: Effective for stable interactions with RIG-I and MAVS

  • Proximity ligation assay: For detecting transient interactions

  • FRET/BRET: For real-time interaction monitoring

  • Cross-linking mass spectrometry: For precise interaction mapping

• Domain-specific considerations:

  • The helicase 1 domain is critical for RIG-I and MAVS interactions

  • The NTR contains a putative double-stranded RNA-binding domain important for RNA interactions

  • The OB domain is essential for interaction with the translation machinery

• Controls for interaction specificity:

  • Domain deletion mutants to map interaction regions

  • Competition assays with purified proteins or domains

  • RNA-dependency tests using RNase treatment

Research has demonstrated that DHX29 interacts with RIG-I and MAVS through its helicase 1 domain, activating the RIG-I–MAVS-dependent cytosolic nucleic acid response pathway .

How might DHX29 be targeted for therapeutic development in viral infections?

Emerging research suggests several approaches for therapeutic targeting of DHX29:

• Potential therapeutic strategies:

  • Small molecule inhibitors targeting DHX29's NTPase activity

  • Peptide inhibitors disrupting specific protein-protein interactions

  • RNA aptamers targeting the RNA-binding domains

• Disease contexts with therapeutic potential:

  • HCMV infection: Recent research shows DHX29 is necessary for efficient translation of HCMV immediate early proteins (IE1/IE2)

  • RNA viral infections: DHX29 contributes to MDA5-mediated EMCV detection

  • Inflammatory conditions: Given DHX29's role in cytokine production

• Considerations for therapeutic development:

  • Cell-type specificity: DHX29's predominant expression in epithelial cells and fibroblasts may allow for targeted approaches

  • Dual function: Separating DHX29's translation and immune functions may be necessary

  • Potential off-target effects: Consider impact on translation of cellular mRNAs with structured 5′ UTRs

Recent studies suggest that "therapies that inhibit DHX29 could potentially be useful in treating HCMV disease and adds to the growing body of literature suggesting DHX29 activity is a disease driver in multiple indications including viral disease, inflammation and cancer" .

What are the current methodological limitations in studying DHX29 and how might they be overcome?

Current research on DHX29 faces several technical challenges:

• Structural complexity limitations:

  • The large size (155 kDa) and complex domain structure make structural studies challenging

  • Solution: Employ cryo-EM approaches and study individual domains separately

• Functional overlap challenges:

  • Difficult to separate translation vs. immune functions

  • Solution: Domain-specific mutations and cell type-specific approaches

• Technical hurdles:

  • Limited availability of well-characterized antibodies

  • Solution: Develop monoclonal antibodies against specific domains

• Methodological innovations needed:

  • Real-time assays to monitor DHX29 activity during scanning

  • Improved methods to study transient interactions during translation initiation

  • Cell-type specific conditional knockout systems

• RNA interaction analysis:

  • Current methods may not capture the full spectrum of RNA targets

  • Solution: Develop CLIP-seq or similar approaches specific for DHX29-RNA interactions

Advancing these methodological approaches will be crucial for fully understanding DHX29's complex roles in both health and disease contexts.