HELZ2 Antibody

What is HELZ2 and why is it significant for research?

HELZ2 (Helicase with Zinc Finger 2, Transcriptional Coactivator) is a multifunctional protein involved in diverse cellular processes, including transcriptional regulation, DNA repair, and cell cycle progression. The significance of HELZ2 in research stems from its roles in several key biological mechanisms:

• It functions as an interferon-stimulated gene (ISG) product with antiviral properties

• It possesses 3'-5' exoribonucleolytic activity despite having a non-consensus residue in its RNB domain active site

• It contains two RNA helicase domains and multiple zinc finger motifs that contribute to its functionality

• It has been implicated in various diseases including viral infections, cancer, and autoimmune disorders

• It plays a role in metabolic regulation as evidenced by knockout studies in mice

Research on HELZ2 has shown its potential involvement in defense mechanisms against viruses such as Dengue virus, hepatitis C virus (HCV), and SARS-CoV-2, making it a protein of interest in immunological and virological research .

What are the common applications for HELZ2 antibodies?

HELZ2 antibodies are primarily used in molecular and cellular biology research applications focused on protein detection and characterization. The main applications include:

• Western Blotting (WB): For detecting and quantifying HELZ2 protein in cell or tissue lysates, typically at recommended dilutions of 1:500-1:2000

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of HELZ2, usually at dilutions around 1:20000

• Immunofluorescence (IF): For visualizing the subcellular localization of HELZ2 in fixed cells

• Immunocytochemistry (ICC): For detecting HELZ2 in cultured cells

Most commercially available HELZ2 antibodies show reactivity against human HELZ2, though some may cross-react with HELZ2 from other species depending on sequence conservation .

What is the difference between various HELZ2 antibody types?

HELZ2 antibodies differ primarily in their targeting region, host species, clonality, and conjugation status:

When selecting an antibody, researchers should consider which isoform of HELZ2 they are targeting, as there are at least two reported isoforms with different molecular weights—HELZ2α (231 kDa) and HELZ2β (295 kDa)—with the latter being the extended N-terminal version .

What are the optimal conditions for Western blot detection of HELZ2?

Detecting HELZ2 via Western blot requires careful optimization due to its large size (approximately 295 kDa for the full-length protein). Recommended protocols include:

• Sample preparation:

  • Use strong lysis buffers containing SDS and protease inhibitors

  • Heat samples at 95°C for 5 minutes to ensure complete denaturation

• Gel electrophoresis:

  • Use low percentage (6-8%) polyacrylamide gels or gradient gels (4-15%)

  • Run at lower voltage (80-100V) for longer periods to achieve better separation of high molecular weight proteins

• Transfer conditions:

  • Wet transfer is preferred over semi-dry for large proteins

  • Transfer overnight at low amperage (30-35 mA) at 4°C for efficient transfer

• Antibody incubation:

  • Recommended dilutions range from 1:500 to 1:2000 for primary antibody

  • Incubate overnight at 4°C for optimal binding

• Detection:

  • Use enhanced chemiluminescence (ECL) or near-infrared fluorescence detection systems

  • Longer exposure times may be necessary due to the size of the protein

When analyzing HELZ2 expression, researchers should be aware that the full-length human HELZ2 protein is longer than previously annotated, as it is translated from a non-canonical start codon extending the protein by 247 residues in Hominidae .

How can I optimize immunofluorescence staining with HELZ2 antibodies?

For successful immunofluorescence staining of HELZ2, consider the following optimization strategies:

• Fixation methods:

  • 4% paraformaldehyde (10-15 minutes at room temperature) for structural preservation

  • Methanol fixation (10 minutes at -20°C) may provide better epitope accessibility

• Permeabilization:

  • 0.1-0.2% Triton X-100 in PBS for 10 minutes

  • Alternative: 0.5% saponin if gentler permeabilization is needed

• Blocking:

  • 5% normal serum (from the same species as the secondary antibody) and 1% BSA in PBS for 1 hour

  • Include 0.1% Triton X-100 in blocking solution to maintain permeabilization

• Antibody dilution:

  • Start with manufacturer's recommended dilution, typically in the range of 1:100-1:500

  • Incubate primary antibody overnight at 4°C for optimal binding

• Signal amplification:

  • Consider tyramide signal amplification if HELZ2 expression is low

  • Alexa Fluor-conjugated HELZ2 antibodies may provide direct detection with reduced background

• Nuclear counterstaining:

  • DAPI or Hoechst for nuclear visualization, as HELZ2 has been reported to have nuclear localization

For co-localization studies, combining HELZ2 staining with stress granule markers like G3BP1 may be informative, especially when studying cellular responses to stress conditions or viral infections .

What controls should be included when working with HELZ2 antibodies?

To ensure experimental validity and interpretable results when working with HELZ2 antibodies, the following controls should be included:

• Positive controls:

  • Cell lines with confirmed HELZ2 expression (examples include various tissues such as heart, pancreas, skeletal muscle, colon, spleen, liver, kidney, lung, peripheral blood, and placenta)

  • Interferon-treated cells, as HELZ2 expression is induced by interferon

• Negative controls:

  • Secondary antibody only (omit primary antibody)

  • Isotype control (irrelevant antibody of the same isotype and host species)

  • HELZ2 knockdown or knockout cells (using siRNA, shRNA, or CRISPR-Cas9)

• Specificity controls:

  • Peptide competition assay using the immunogen peptide

  • Validation across multiple applications (e.g., IF results should align with WB results)

  • Detection of expected molecular weight band (approximately 295 kDa for full-length HELZ2)

• Expression modulation controls:

  • Interferon treatment (should increase HELZ2 expression)

  • Viral infection models, which may alter HELZ2 expression depending on the virus

• Isoform controls:

  • When possible, distinguish between HELZ2α (231 kDa) and HELZ2β (295 kDa) using antibodies targeting specific regions

It's important to note that HELZ2 expression can be modulated by interferon treatment, which provides a useful positive control condition for antibody validation experiments .

How can HELZ2 antibodies be used to study its role in antiviral immunity?

HELZ2 antibodies can be instrumental in investigating its role in antiviral immunity through several methodological approaches:

• Expression analysis during viral infection:

  • Use Western blot with HELZ2 antibodies to quantify expression levels in response to different viruses (HELZ2 is upregulated following SARS-CoV-2, Dengue virus, and HCV infections)

  • Examine temporal expression patterns using time-course experiments to determine when HELZ2 is induced post-infection

• Subcellular localization studies:

  • Employ immunofluorescence to track HELZ2 localization during viral infection

  • Co-localization studies with viral components to identify potential interaction sites

  • Investigation of stress granule association using markers like G3BP1

• Protein-protein interaction studies:

  • Immunoprecipitation with HELZ2 antibodies followed by mass spectrometry to identify viral or host interaction partners

  • Co-immunoprecipitation to confirm specific interactions with viral components

• Functional studies:

  • Combine HELZ2 antibodies with RNA-immunoprecipitation (RNA-IP) to identify target RNAs, particularly viral RNAs

  • Use HELZ2 antibodies to correlate protein levels with functional readouts like viral replication efficiency or interferon signaling

  • Monitor HELZ2 degradation of structured RNAs using its 3'-5' exoribonucleolytic activity

• Manipulation experiments:

  • Compare viral replication in HELZ2 knockdown/knockout cells versus controls

  • Rescue experiments using wild-type versus mutant HELZ2 (e.g., mutations that abolish RNase activity)

Research has demonstrated that HELZ2 mediates inhibition of Dengue virus in human hepatic cells induced by IFN-α and reduces the antiviral effects against HCV when downregulated. It has also been implicated in SARS-CoV-2 infection, potentially having a proviral effect, highlighting the complexity of its roles across different viral infections .

What approaches can be used to study HELZ2's enzymatic activities?

Studying HELZ2's enzymatic activities requires specialized experimental approaches targeting its 3'-5' exoribonuclease and helicase functions:

• Exoribonuclease activity assays:

  • Use radiolabeled or fluorescently labeled RNA substrates incubated with immunopurified HELZ2

  • Analyze degradation products by gel electrophoresis

  • Compare wild-type HELZ2 with mutants containing substitutions in key residues of the RNB domain

  • Include controls to distinguish 3'-5' vs. 5'-3' degradation (using RNA with protected ends)

• RNA helicase activity assays:

  • Design RNA duplexes with single-stranded overhangs

  • Monitor unwinding using FRET-based assays or gel-based methods

  • Assess ATP dependence by comparing activity with and without ATP

  • Evaluate synergy between helicase and RNase domains using structured RNA substrates

• Specificity determinations:

  • CLIP-seq (Cross-linking immunoprecipitation followed by sequencing) using HELZ2 antibodies to identify RNA binding sites in vivo

  • RNA structure mapping of preferred substrates

  • Competition assays with structured vs. unstructured RNAs

• Kinetic analyses:

  • Determine reaction rates under different conditions (temperature, pH, ion concentrations)

  • Analyze the effects of ATP concentration on helicase activity

  • Investigate the processivity of HELZ2 exoribonuclease activity

• Validation in cellular contexts:

  • Assess degradation of specific RNAs (e.g., LINE-1 RNA) in cells expressing wild-type vs. enzymatically inactive HELZ2

  • Monitor effects on reporter genes containing putative HELZ2 target sequences

Recent research has shown that HELZ2 is particularly effective at degrading structured RNAs through the coordinated action of its ATP-dependent helicase domains and 3'-5' ribonucleolytic activity, a property that may be crucial for targeting viral RNAs or mobile genetic elements like LINE-1 .

How can HELZ2 antibodies be used to investigate its role in cancer and autoimmune diseases?

HELZ2 antibodies can facilitate research into its roles in cancer and autoimmune diseases through several experimental approaches:

• Expression profiling in disease contexts:

  • Immunohistochemistry of tissue microarrays from cancer patients or autoimmune disease samples

  • Western blot analysis comparing HELZ2 levels in normal vs. diseased tissues

  • Correlation of HELZ2 expression with disease progression or patient outcomes

• Mutation analysis:

  • Immunoprecipitation followed by mass spectrometry to identify post-translational modifications in disease states

  • Combining HELZ2 antibody detection with sequencing to correlate protein expression with specific mutations

  • Functional studies of cancer-associated HELZ2 mutants to assess effects on RNase activity

• Autoimmune disease investigations:

  • Analysis of HELZ2 antibody reactivity in patient samples to investigate if HELZ2 itself becomes an autoantigen

  • Immunoprecipitation studies to identify disease-specific interaction partners

  • Examination of HELZ2 function in immune cells from patients with autoimmune conditions

• Signaling pathway analysis:

  • Phospho-specific antibodies (if available) to monitor HELZ2 activation status

  • Co-immunoprecipitation studies to map HELZ2 interactome in normal vs. cancer cells

  • Analysis of HELZ2's impact on known oncogenic or tumor-suppressive pathways

• Therapeutic response studies:

  • Monitoring HELZ2 expression during treatment with immunotherapies or other cancer therapies

  • Assessing whether HELZ2 status predicts response to interferon-based therapies

Research has identified that HELZ2 somatic mutations are frequently found in cancer patients, with some mutations abolishing its RNase activity. Additionally, genetic association studies have linked HELZ2 locus variants with autoimmune diseases, particularly an autoimmune disease locus in B cells and primary biliary cholangitis, an autoimmune liver disease .

Why might I detect multiple bands when using HELZ2 antibodies in Western blot?

Multiple bands in Western blot using HELZ2 antibodies can occur for several biological and technical reasons:

• Isoform detection:

  • HELZ2 has at least two documented isoforms: HELZ2α (231 kDa) and HELZ2β (295 kDa)

  • Additional alternative splicing events may generate further isoforms

  • Solution: Use isoform-specific antibodies when available, or compare with known molecular weight markers

• Post-translational modifications:

  • Phosphorylation, ubiquitination, or other modifications can alter migration patterns

  • Solution: Use phosphatase treatment or other enzymatic treatments to confirm modification status

• Protein degradation:

  • HELZ2's large size makes it susceptible to proteolytic degradation during sample preparation

  • Solution: Use fresh samples, keep them cold, include additional protease inhibitors, and avoid freeze-thaw cycles

• Non-canonical translation:

  • Research has revealed that human HELZ2 is translated from a non-canonical start codon in Hominidae, extending the protein by 247 residues

  • Solution: Confirm band sizes against positive controls from well-characterized cell lines

• Cross-reactivity:

  • Antibodies may detect related proteins with similar epitopes

  • Solution: Validate with HELZ2 knockdown/knockout samples or peptide competition assays

• Technical issues:

  • Insufficient blocking or high antibody concentration can lead to non-specific binding

  • Solution: Optimize blocking conditions and antibody dilutions

When analyzing HELZ2 expression, researchers should be aware that the full-length human HELZ2 protein migrates at approximately 295 kDa, though exact migration patterns may vary depending on the gel system used .

How can I distinguish between specific and non-specific staining when using HELZ2 antibodies for immunofluorescence?

Distinguishing specific from non-specific staining in immunofluorescence requires careful experimental design and appropriate controls:

• Validation controls:

  • HELZ2 knockdown/knockout cells as negative controls

  • Interferon-treated cells as positive controls (HELZ2 is interferon-inducible)

  • Secondary antibody only controls to assess background fluorescence

  • Peptide competition assays to confirm epitope specificity

• Signal evaluation criteria:

  • Subcellular localization consistency with reported HELZ2 localization (primarily nuclear)

  • Staining pattern correlation with HELZ2 expression modulation (e.g., increased after interferon treatment)

  • Consistency across different fixation and permeabilization methods

  • Reproducibility across different samples and experiments

• Technical optimization:

  • Titration of primary antibody to determine optimal concentration

  • Comparison of different fixation methods (paraformaldehyde vs. methanol)

  • Extended washing steps to reduce background

  • Use of specialized blocking agents to reduce non-specific binding

• Advanced validation approaches:

  • Dual labeling with two different HELZ2 antibodies targeting different epitopes

  • Correlation of immunofluorescence results with other detection methods (Western blot, RNA expression)

  • Super-resolution microscopy to confirm subcellular localization details

• Signal amplification considerations:

  • If using signal amplification methods, include appropriate controls for each step

  • With directly conjugated antibodies, ensure fluorophore doesn't affect antibody binding

Research indicates that HELZ2 can form cytoplasmic foci under certain conditions, particularly in response to stress or viral infection, which may complicate the interpretation of immunofluorescence results. When studying such phenomena, co-localization with known stress granule markers like G3BP1 can help confirm the specificity of the observed patterns .

What factors should be considered when interpreting changes in HELZ2 expression in experimental studies?

• Interferon response effects:

  • HELZ2 is an interferon-stimulated gene, so changes may reflect broader interferon responses rather than direct experimental effects

  • Solution: Include interferon neutralizing antibodies as controls or measure other ISGs concurrently

• Viral infection considerations:

  • Different viruses affect HELZ2 expression differently (e.g., upregulation with SARS-CoV-2, Dengue virus)

  • Solution: Time course experiments to distinguish primary vs. secondary effects

• Cell type specificity:

  • HELZ2 expression and function may vary across cell types

  • Solution: Validate findings across multiple cell lines or primary cells relevant to research question

• Isoform-specific regulation:

  • Expression changes may affect specific isoforms differently

  • Solution: Use isoform-specific detection methods when possible

• Post-translational regulation:

  • Protein levels may not correlate with activity due to post-translational modifications

  • Solution: Combine expression analysis with functional assays

• Technical considerations:

  • Large proteins like HELZ2 may transfer inefficiently in Western blotting

  • Solution: Use appropriate controls for protein loading and transfer efficiency

• Feedback mechanisms:

  • HELZ2 itself may affect interferon responses, creating complex feedback loops

  • Solution: Use HELZ2 mutants to dissect specific functions

Research has shown that HELZ2 can both respond to and modulate interferon responses, particularly in the context of viral infections. Additionally, HELZ2 has been shown to inhibit retrotransposition of human LINE-1 elements and can reduce the type I interferon response associated with LINE-1 expression, suggesting complex regulatory relationships .

What emerging techniques might enhance HELZ2 antibody applications in research?

Several emerging techniques promise to expand the utility of HELZ2 antibodies in research:

• Proximity labeling approaches:

  • BioID or APEX2 fusion to HELZ2 combined with antibody detection to map its dynamic interactome

  • TurboID for faster labeling in time-sensitive processes like viral infection

  • Significance: Will reveal transient interactions missed by conventional co-immunoprecipitation

• Advanced imaging techniques:

  • Live-cell nanobody-based tracking of HELZ2 dynamics during viral infection

  • Super-resolution microscopy combined with HELZ2 antibodies for precise localization

  • Lattice light-sheet microscopy for 3D visualization of HELZ2 interactions

  • Significance: Will provide spatial and temporal resolution of HELZ2 function

• Single-cell applications:

  • Combining HELZ2 immunostaining with single-cell RNA-seq for correlative analysis

  • CITE-seq or REAP-seq to simultaneously measure HELZ2 protein and transcript levels

  • Significance: Will reveal cell-to-cell variability in HELZ2 expression and function

• CRISPR-based approaches:

  • CRISPR activation/inhibition systems combined with HELZ2 antibody detection

  • Endogenous tagging of HELZ2 for live-cell imaging and functional studies

  • Significance: Will allow precise manipulation of HELZ2 expression

• Structural biology applications:

  • Cryo-electron microscopy of HELZ2 complexes isolated with specific antibodies

  • Hydrogen-deuterium exchange mass spectrometry combined with epitope-specific antibodies

  • Significance: Will provide structural insights into HELZ2 function

These emerging techniques will be particularly valuable for investigating HELZ2's role in coordinating ATP-dependent RNA helicase activity with 3'-5' exoribonucleolytic function to degrade structured RNAs, a property that may be crucial for its antiviral and anti-retrotransposon activities .

What are the key unresolved questions about HELZ2 function that antibody-based approaches could help address?

Several critical unresolved questions about HELZ2 function could be addressed using antibody-based approaches:

• Substrate specificity mechanisms:

  • Question: How does HELZ2 select its RNA targets for degradation?

  • Approach: CLIP-seq using HELZ2 antibodies to map binding sites on endogenous RNAs

  • Significance: Will reveal recognition motifs or structures that determine HELZ2 activity

• Regulatory pathways:

  • Question: Beyond interferon, what signals regulate HELZ2 expression and activity?

  • Approach: Antibody-based phospho-proteomics to identify post-translational modifications

  • Significance: Will identify additional regulatory pathways controlling HELZ2

• Functional domains interplay:

  • Question: How do the helicase and RNase domains coordinate their activities?

  • Approach: Conformation-specific antibodies to detect structural changes during activation

  • Significance: Will help design specific inhibitors or activators of HELZ2

• Viral counterstrategies:

  • Question: Do viruses encode proteins that specifically inhibit HELZ2?

  • Approach: Immunoprecipitation followed by mass spectrometry in infected cells

  • Significance: Will identify potential viral antagonists of this host defense mechanism

• Therapeutic potential:

  • Question: Could modulation of HELZ2 activity be therapeutically beneficial?

  • Approach: Correlation of HELZ2 levels (detected by immunohistochemistry) with disease outcomes

  • Significance: Will guide development of HELZ2-targeted therapeutics

• Isoform-specific functions:

  • Question: Do HELZ2α and HELZ2β isoforms have distinct functions?

  • Approach: Isoform-specific antibodies combined with functional assays

  • Significance: Will clarify the biological significance of alternative translation start sites

Research has already established that HELZ2 plays roles in antiviral immunity, metabolic regulation, and potentially cancer suppression, but the molecular mechanisms underlying these diverse functions remain incompletely understood. Antibody-based approaches will be critical for dissecting these mechanisms .

How can HELZ2 antibodies be used to study its role in metabolic regulation?

HELZ2 antibodies can be employed to investigate its role in metabolic regulation through several experimental approaches:

• Tissue-specific expression analysis:

  • Immunohistochemistry of metabolic tissues (liver, adipose, muscle) in normal vs. high-fat diet conditions

  • Western blot analysis comparing HELZ2 levels across metabolic states (fed, fasted, insulin-stimulated)

  • Significance: Will establish tissue-specific regulation patterns related to metabolic conditions

• Transcriptional complex analysis:

  • Co-immunoprecipitation with HELZ2 antibodies to identify interactions with nuclear receptors (PPARα, PPARγ)

  • ChIP-seq to map HELZ2 binding sites on regulatory regions of metabolic genes

  • Significance: Will define HELZ2's role as a transcriptional coactivator for metabolic gene regulation

• Metabolic challenge studies:

  • Tracking HELZ2 expression and localization during metabolic stresses (glucose deprivation, lipid loading)

  • Correlating HELZ2 levels with metabolic parameters in intervention studies

  • Significance: Will reveal dynamic regulation of HELZ2 in response to metabolic changes

• Post-translational modification analysis:

  • Phospho-specific antibodies to monitor HELZ2 activation status in response to insulin or other metabolic signals

  • Analysis of other modifications (acetylation, SUMOylation) that might regulate HELZ2 activity

  • Significance: Will identify regulatory mechanisms controlling HELZ2 function

• Leptin signaling studies:

  • Co-localization studies with leptin receptor in liver and brain tissues

  • Immunoprecipitation to detect HELZ2-leptin receptor complexes

  • Significance: Will clarify HELZ2's role in leptin signaling, which was implicated by knockout studies

Research with HELZ2-knockout mice has demonstrated that these animals are resistant to high-fat diet-induced obesity, glucose intolerance, and hepatosteatosis, indicating a critical role for HELZ2 in metabolic regulation. These phenotypes were attributed to central leptin resistance and increased leptin receptor mRNA in the liver, suggesting complex tissue-specific roles for HELZ2 in metabolism .

What controls should be included when studying HELZ2's interaction with nuclear receptors?

When investigating HELZ2's interactions with nuclear receptors such as PPARα and PPARγ, researchers should include several specialized controls:

• Ligand-dependent interaction controls:

  • Compare interactions in the presence vs. absence of receptor-specific ligands

  • Include antagonists to confirm specificity of ligand effects

  • Test physiologically relevant concentrations and time courses

  • Significance: Will distinguish constitutive from ligand-induced interactions

• Domain-specific interaction controls:

  • Use truncated versions of HELZ2 to map interaction domains

  • Include mutations in the LXXLL nuclear receptor interaction motifs of HELZ2

  • Test DNA-binding domain and ligand-binding domain constructs of nuclear receptors separately

  • Significance: Will define the molecular basis of interactions

• Transcriptional activity controls:

  • Correlate protein-protein interactions with functional readouts (reporter gene assays)

  • Include transcriptionally inactive receptor mutants

  • Test effects of HELZ2 RNase activity mutations on transcriptional outcomes

  • Significance: Will link physical interactions to functional consequences

• Competitive binding controls:

  • Include known coactivators (p300, SRC-1) in competition experiments

  • Test effects of other nuclear receptors on HELZ2-PPAR interactions

  • Use peptide competitors based on interaction domains

  • Significance: Will establish specificity and relative affinity of interactions

• Cell type-specific controls:

  • Compare interactions in metabolically relevant cell types (hepatocytes, adipocytes)

  • Test effects of differentiation state on interaction strength

  • Include cell types with different HELZ2 isoform expression patterns

  • Significance: Will reveal context-dependent regulatory mechanisms