ZDHHC23 Antibody

What is ZDHHC23 and what are its primary cellular functions?

ZDHHC23 belongs to the zinc finger DHHC domain-containing protein family, known primarily for their palmitoyltransferase (PAT) activity. This enzyme catalyzes protein S-palmitoylation, a post-translational modification that adds palmitate to specific cysteine residues of target proteins . ZDHHC23 plays crucial roles in diverse cellular processes, with recent research highlighting its importance in:

• Immune regulation, particularly in teleost immune responses

• Modulation of inflammatory responses

• Promotion of M2-type macrophage polarization

• Inhibition of macrophage necroptosis during bacterial infection

• Potential involvement in cytoskeletal reorganization and adhesion pathways

In neuroblastoma, ZDHHC23 has been identified as a potential biomarker correlated with poor patient prognosis, showing significant upregulation under hypoxic conditions (1% O₂) compared to normoxic conditions (21% O₂) . This oxygen-dependent regulation suggests ZDHHC23 may play important roles in cellular adaptation to hypoxic environments.

How does ZDHHC23 contribute to immune regulation?

ZDHHC23 exhibits significant anti-inflammatory properties in immune cells. Research in teleost models demonstrates that ZDHHC23 silencing leads to heightened pro-inflammatory cytokine expression (IL-1β, IL-6) and diminished anti-inflammatory cytokine levels (IL-10, TGF-β) during bacterial infection . This indicates ZDHHC23 serves as a negative regulator of inflammatory responses.

Mechanistically, ZDHHC23 promotes M2-type macrophage polarization while inhibiting M1-type polarization, as evidenced by:

• Increased expression of M1 markers (CXCL9, iNOS) in ZDHHC23-knockdown macrophages

• Decreased expression of M2 markers in ZDHHC23-deficient cells

• Enhanced phagocytic activity in ZDHHC23-knockdown macrophages

Additionally, ZDHHC23 facilitates necroptosis in infected macrophages, as demonstrated by delayed and reduced phosphorylation of necroptosis markers (RIP1, RIP3, MLKL) upon ZDHHC23 knockdown . This suggests ZDHHC23 may help resolve infections by promoting controlled cell death of infected immune cells.

What distinguishes ZDHHC23 from other ZDHHC family members?

While all ZDHHC family members contain a highly conserved DHHC domain responsible for palmitoyltransferase activity, ZDHHC23 exhibits several distinguishing characteristics:

• Immune regulation: ZDHHC23 shows notable upregulation following bacterial infection, with demonstrated anti-inflammatory properties. Unlike ZDHHC1 and ZDHHC11, which function as positive regulators of DNA virus-triggered signaling, ZDHHC23 appears to dampen inflammatory responses .

• Oxygen-sensitive interactions: ZDHHC23's interactome changes dramatically (~70% of binding partners) between normoxic and hypoxic conditions, suggesting unique oxygen-sensitive regulatory mechanisms .

• Disease associations: In neuroblastoma, ZDHHC23 expression correlates strongly with poor prognosis under hypoxic conditions, identifying it as a potential biomarker for aggressive disease .

• Structural features: All ZDHHC23 proteins exhibit a highly conserved DHHC domain across species, confirmed by tertiary structure prediction, but may have unique regulatory domains that differentiate their function .

What challenges exist in developing specific antibodies against ZDHHC23?

Developing specific antibodies against ZDHHC23 presents several significant challenges that researchers should be aware of:

• Limited antibody specificity: Commercial antibodies for ZDHHC23 have demonstrated poor specificity for both immunostaining and immunoblotting of endogenous protein. Research indicates these antibodies often lack sufficient specificity for reliable detection .

• Low endogenous expression: ZDHHC23 may be expressed at relatively low levels in many cell types, making detection of endogenous protein challenging even with targeted mass spectrometry approaches. Researchers have reported difficulties in detecting endogenous ZDHHC23 even with Parallel Reaction Monitoring (PRM) techniques .

• Transmembrane protein challenges: As ZDHHC23 is predicted to be a multi-pass membrane protein, certain epitopes may be inaccessible to antibodies due to membrane integration, requiring specialized extraction methods.

• Cross-reactivity with other ZDHHC family members: The conserved DHHC domain across family members increases the risk of antibody cross-reactivity, necessitating careful validation.

To overcome these challenges, researchers have employed alternative strategies including:

• Development of epitope-tagged ZDHHC23 constructs (e.g., HA-mCherry-ZDHHC23) for immunoprecipitation and localization studies

• Optimization of mass spectrometry protocols for protein detection

• Development of RT-qPCR assays to reliably quantify ZDHHC23 mRNA expression

What strategies are most effective for detecting and studying ZDHHC23 in experimental settings?

Based on current research, several strategies have proven effective for studying ZDHHC23:

• Expression constructs with dual reporters:

  • HA-mCherry-ZDHHC23 or ZDHHC23-mCherry-HA constructs facilitate both visualization (via mCherry) and immunoprecipitation (via HA tag)

  • Testing both N-terminal and C-terminal tagging approaches is advisable as tag position may affect protein function

• RT-qPCR for expression analysis:

  • Reliable detection of ZDHHC23 mRNA using validated primers

  • Normalization against appropriate housekeeping genes (e.g., 18S rRNA)

  • Triplicate reactions with multiple biological replicates

• Immunoprecipitation for interaction studies:

  • HA-tag based immunoprecipitation protocols optimized for LC-MS/MS analysis

  • Stringent washing conditions to minimize non-specific binding

  • Parallel immunoprecipitations under different oxygen tensions (21% vs. 1% O₂) to capture oxygen-dependent interactions

• Click chemistry for palmitoylation targets:

  • Metabolic labeling with azide-palmitic acid followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC)

  • Optimization of azide-palmitic acid concentration for efficient labeling

  • Comparison between wild-type ZDHHC23 and catalytically inactive mutants (ZDHHC23 to ZDHHS23)

• Functional assays:

  • siRNA-mediated knockdown followed by assessment of:

    • Cytokine expression profiles

    • Macrophage polarization markers

    • Phagocytic activity

    • Necroptosis markers (phosphorylation of RIP1, RIP3, MLKL)

These methodologies should be adapted to specific research questions and cellular contexts, with careful consideration of appropriate controls in each experimental setting.

How can researchers optimize protein interaction studies for ZDHHC23?

Optimizing protein interaction studies for ZDHHC23 requires careful consideration of several key factors:

• Extraction and solubilization conditions:

  • Use of appropriate detergents (e.g., 1% NP-40, 0.5% Triton X-100) to efficiently solubilize membrane-associated ZDHHC23 while preserving protein-protein interactions

  • Inclusion of protease inhibitors to prevent degradation

  • Optimization of buffer conditions (salt concentration, pH) to maintain interactions

• Immunoprecipitation strategy:

  • Expression of epitope-tagged ZDHHC23 (HA-tag recommended based on published protocols)

  • Optimization of antibody-to-bead ratio and incubation conditions

  • Stringent washing to remove non-specific binding without disrupting true interactions

• Oxygen-dependent interactions:

  • Parallel immunoprecipitations under both normoxic (21% O₂) and hypoxic (1% O₂) conditions

  • Rapid sample processing to prevent reoxygenation artifacts

  • Quantitative comparison of interaction partners between conditions

• Mass spectrometry analysis:

  • Label-free quantification for relative abundance of interaction partners

  • Multiple biological replicates (minimum of three) to identify consistent interactions

  • Statistical analysis to distinguish specific interactions from background

• Validation approaches:

  • Reciprocal co-immunoprecipitation of key interaction partners

  • Proximity ligation assays to confirm interactions in intact cells

  • Functional validation through siRNA knockdown of interaction partners

Research has shown that approximately 70% of ZDHHC23's interactome changes between normoxic and hypoxic conditions, highlighting the importance of controlling oxygen tension during interaction studies . GO-term analysis of these interactomes suggests that ZDHHC23 is a component of several potentially important pathways, including cytoskeletal reorganization and adhesion .

How can researchers differentiate between palmitoyltransferase-dependent and independent functions of ZDHHC23?

Distinguishing between functions that depend on ZDHHC23's palmitoyltransferase (PAT) activity versus those mediated by protein-protein interactions requires sophisticated experimental approaches:

• Generation of catalytically inactive mutants:

  • Creation of point mutations in the DHHC domain (e.g., DHHC to DHHS) that abolish enzymatic activity

  • Validation of PAT activity loss using click chemistry with azide-palmitic acid

  • Comparison of wild-type versus mutant ZDHHC23 in functional assays

• Complementation experiments:

  • Knockdown or knockout of endogenous ZDHHC23

  • Rescue with either wild-type or catalytically inactive ZDHHC23

  • Assessment of which phenotypes are rescued by each construct

• Domain mapping:

  • Generation of truncation mutants maintaining the DHHC domain but lacking other regions

  • Systematic assessment of which protein interactions and functions are preserved

  • Identification of domains responsible for PAT-independent functions

• Substrate-specific approaches:

  • Identification of ZDHHC23 palmitoylation targets using proteomics

  • Mutation of palmitoylation sites in key substrates (Cys to Ser/Ala)

  • Determination of whether non-palmitoylatable substrate mutants phenocopy ZDHHC23 loss

• Comparative interactome analysis:

  • Comparison of protein interaction networks between wild-type and catalytically inactive ZDHHC23

  • Identification of interactions that persist despite loss of PAT activity

  • Network analysis to reveal potential PAT-independent signaling pathways

Research indicates that several ZDHHC family members, including ZDHHC1 and ZDHHC11, have documented non-PAT functions in immune regulation . Similar approaches could reveal whether ZDHHC23's roles in macrophage polarization and necroptosis regulation depend on its enzymatic activity or represent novel non-PAT functions.

What methodologies are most effective for studying ZDHHC23's role in macrophage polarization?

Investigating ZDHHC23's role in macrophage polarization requires multifaceted approaches to capture both molecular mechanisms and functional outcomes:

• Polarization marker analysis:

  • Assessment of M1 markers (CXCL9, iNOS) and M2 markers in ZDHHC23-manipulated macrophages

  • Flow cytometry for surface marker expression

  • RT-qPCR and ELISA for cytokine profiles (IL-1β, IL-6, IL-10, TGF-β)

• Functional characterization:

  • Phagocytic activity assays using fluorescently labeled particles or bacteria

  • ROS production measurement

  • Migration and chemotaxis assays

  • Bacterial killing capacity assessment

• Signaling pathway analysis:

  • Phosphorylation status of key polarization regulators (STAT1/STAT6, NF-κB)

  • Inhibitor studies to identify critical signaling nodes

  • Temporal analysis of signaling events following polarization stimuli

• Gene expression profiling:

  • RNA-seq comparing wild-type vs. ZDHHC23-deficient macrophages under M1/M2 polarizing conditions

  • ChIP-seq to identify transcription factors regulated by ZDHHC23

  • Integration with public datasets on macrophage polarization

• Target identification and validation:

  • Palmitoylation proteomics to identify ZDHHC23 substrates in macrophages

  • Focus on proteins involved in polarization pathways

  • Site-directed mutagenesis of palmitoylation sites in key targets

Research has demonstrated that silencing ZDHHC23 significantly skews macrophages toward a pro-inflammatory M1 phenotype during bacterial infection, with increased expression of pro-inflammatory cytokines and decreased anti-inflammatory cytokine production . This indicates ZDHHC23 plays a critical role in promoting M2-type polarization, potentially as a mechanism to resolve inflammation and prevent excessive tissue damage during infection.

How can researchers investigate ZDHHC23 function under different oxygen tensions?

Studying ZDHHC23 under different oxygen tensions requires specialized approaches to capture oxygen-dependent changes in protein function:

• Controlled oxygen environments:

  • Use of hypoxia chambers or workstations that maintain precise O₂ levels (e.g., 1% for hypoxia, 21% for normoxia)

  • Implementation of rapid sample processing to prevent reoxygenation artifacts

  • Consideration of intermediate O₂ levels to establish dose-response relationships

• Comparative interactome analysis:

  • Parallel immunoprecipitation experiments under different O₂ tensions

  • Quantitative proteomics to compare interaction partners

  • Network analysis to identify oxygen-sensitive interaction hubs

• Palmitoylation target identification:

  • Click chemistry with azide-palmitic acid under different O₂ tensions

  • Comparison of palmitoylated targets to distinguish O₂-independent vs. O₂-sensitive targets

  • Validation of key targets using site-directed mutagenesis

• Functional studies:

  • Assessment of ZDHHC23 enzymatic activity under different O₂ tensions

  • Examination of subcellular localization changes using live-cell imaging

  • Determination of whether ZDHHC23 undergoes post-translational modifications in response to O₂ changes

• Integration with hypoxia response pathways:

  • Investigation of potential interactions between ZDHHC23 and hypoxia-inducible factors (HIFs)

  • Assessment of whether ZDHHC23 is a direct HIF target gene

  • Determination of ZDHHC23's contribution to cellular adaptation to hypoxia

Research has demonstrated that approximately 70% of ZDHHC23's interactome changes between normoxic and hypoxic conditions, highlighting the significant impact of oxygen tension on ZDHHC23 function . GO-term analysis of these oxygen-dependent interactions suggests involvement in cytoskeletal reorganization and adhesion pathways, which may be particularly relevant in hypoxic tumor microenvironments.

What are common technical challenges when working with ZDHHC23 antibodies?

Working with ZDHHC23 antibodies presents several technical challenges that researchers should anticipate and address:

• Limited specificity:

  • Commercial antibodies for ZDHHC23 have demonstrated poor specificity for both immunostaining and immunoblotting of endogenous protein

  • Recommendation: Always include appropriate controls (ZDHHC23 knockdown/knockout) to confirm specificity

  • Consider using epitope-tagged ZDHHC23 constructs when possible

• Low endogenous expression:

  • ZDHHC23 may be expressed at relatively low levels in many cell types

  • Strategy: Use signal amplification methods (e.g., TSA for immunohistochemistry)

  • Consider concentrating protein samples for Western blotting

  • Implement immunoprecipitation before detection to enrich the target protein

• Cross-reactivity with other ZDHHC family members:

  • The conserved DHHC domain may lead to antibody cross-reactivity

  • Solution: Use antibodies raised against unique regions of ZDHHC23

  • Validate using cells expressing different ZDHHC family members

• Membrane protein solubilization issues:

  • As a predicted multi-pass membrane protein, ZDHHC23 may require optimization of extraction conditions

  • Try different detergents (NP-40, Triton X-100, CHAPS) at varying concentrations

  • Consider membrane fractionation approaches to enrich for ZDHHC23

• Detection sensitivity limitations:

  • Even targeted mass spectrometry approaches (PRM) have shown limitations in detecting endogenous ZDHHC23

  • Consider using more sensitive detection methods or cellular models with higher expression

When antibody-based detection proves challenging, researchers should complement with alternative approaches such as RT-qPCR, fluorescently tagged expression constructs, or targeted mass spectrometry to provide corroborating evidence for ZDHHC23 expression and function.

What controls are essential when conducting ZDHHC23 antibody-based experiments?

Implementing appropriate controls is crucial for generating reliable data with ZDHHC23 antibodies:

• Specificity controls:

  • ZDHHC23 knockdown or knockout samples as negative controls

  • Overexpression of ZDHHC23 as positive controls

  • Peptide competition assays to confirm epitope specificity

  • Comparison with alternative detection methods (e.g., RT-qPCR)

• Loading and transfer controls:

  • Use of housekeeping proteins (β-actin, GAPDH) for Western blotting

  • Ponceau S staining of membranes to confirm protein transfer

  • Inclusion of recombinant ZDHHC23 protein as a reference standard

• Immunoprecipitation controls:

  • IgG control immunoprecipitations to identify non-specific binding

  • Pre-clearing lysates to reduce background

  • Input samples (typically 5-10% of immunoprecipitation volume)

  • Reverse immunoprecipitation to confirm interactions

• Immunofluorescence controls:

  • Secondary antibody-only controls to assess background

  • Blocking peptide controls to confirm specificity

  • Co-localization with organelle markers to confirm subcellular distribution

• Experimental validation controls:

  • Multiple siRNAs targeting different regions of ZDHHC23 to control for off-target effects

  • Rescue experiments with siRNA-resistant ZDHHC23 constructs

  • Comparison between wild-type and catalytically inactive ZDHHC23 mutants

• Oxygen tension controls:

  • When studying oxygen-dependent effects, maintain strict control of O₂ levels

  • Include hypoxia marker controls (e.g., HIF-1α stabilization)

  • Process samples rapidly to prevent reoxygenation artifacts

Implementation of these controls is essential for generating reproducible and reliable data, particularly given the documented challenges with ZDHHC23 antibody specificity.

How can researchers design optimal ZDHHC23 expression constructs for antibody validation and functional studies?

Designing effective ZDHHC23 expression constructs requires careful consideration of several factors:

• Tag selection and positioning:

  • Dual reporter systems (e.g., HA-mCherry-ZDHHC23 or ZDHHC23-mCherry-HA) facilitate both visualization and immunoprecipitation

  • Consider testing both N-terminal and C-terminal tagging approaches, as tag position may affect protein function

  • Small epitope tags (HA, FLAG, Myc) are preferable for immunoprecipitation

  • Fluorescent protein tags (mCherry, GFP) enable live-cell visualization

• Vector selection:

  • Choose vectors with appropriate promoters for the target cell type

  • Consider inducible expression systems for temporal control

  • Ensure vector compatibility with experimental goals (transient vs. stable expression)

• Codon optimization:

  • Optimize codons for the expression system being used

  • Consider species-specific codon preferences if working with cross-species models

• Mutagenesis strategies:

  • Design catalytically inactive mutants (DHHC to DHHS) for distinguishing enzymatic vs. structural roles

  • Create domain deletion constructs to map functional regions

  • Consider palmitoylation site mutants of ZDHHC23 itself if it undergoes auto-palmitoylation

• Cloning strategy optimization:

  • Use seamless cloning methods to avoid introducing additional sequences

  • Verify constructs by sequencing the entire coding region

  • Confirm expression by Western blotting before functional studies

• Expression validation:

  • Verify correct subcellular localization of tagged proteins

  • Compare expression levels to endogenous protein where possible

  • Confirm functionality of tagged constructs in rescue experiments

Successful ZDHHC23 construct design has been demonstrated using PCR amplification of the full-length open reading frame followed by seamless cloning into expression vectors containing appropriate tags . These constructs can serve as valuable positive controls for antibody validation and as tools for investigating ZDHHC23 function in various experimental contexts.

What are emerging areas of investigation for ZDHHC23 antibody applications?

Several promising research directions are emerging for ZDHHC23 antibody applications:

• Single-cell analysis:

  • Developing antibodies suitable for single-cell proteomics

  • Investigating cell-to-cell variability in ZDHHC23 expression and localization

  • Integrating with single-cell transcriptomics to correlate protein and mRNA levels

• Proximity labeling approaches:

  • Engineering ZDHHC23 fusion proteins with proximity labeling enzymes (BioID, APEX2)

  • Mapping the spatial proteome surrounding ZDHHC23 in different subcellular compartments

  • Capturing transient interactions that may be lost in conventional immunoprecipitation

• In vivo imaging:

  • Developing antibody-based probes for in vivo imaging of ZDHHC23 in disease models

  • Creating high-affinity recombinant antibody fragments optimized for tissue penetration

  • Multiplexed imaging to correlate ZDHHC23 with other disease markers

• Therapeutic targeting:

  • Developing function-blocking antibodies against ZDHHC23 for potential therapeutic applications

  • Creating antibody-drug conjugates targeting cells with high ZDHHC23 expression

  • Engineering bispecific antibodies to modulate ZDHHC23 activity in specific cellular contexts

• Structural biology applications:

  • Using antibodies as crystallization chaperones for structural studies of ZDHHC23

  • Developing conformation-specific antibodies to capture different functional states

  • Cryo-EM studies of ZDHHC23 complexes facilitated by antibody binding

As ZDHHC23 emerges as a potential biomarker in diseases like neuroblastoma and a regulator of immune responses , these advanced antibody applications could significantly advance our understanding of its functions and therapeutic potential.

How might novel antibody technologies improve ZDHHC23 detection and characterization?

Emerging antibody technologies offer promising approaches to overcome current limitations in ZDHHC23 research:

• Recombinant antibody engineering:

  • Development of high-affinity recombinant antibodies through display technologies (phage, yeast, mammalian)

  • Engineering of single-domain antibodies (nanobodies) with enhanced access to sterically hindered epitopes

  • Creation of bispecific antibodies targeting multiple regions of ZDHHC23 for improved specificity

• Advanced imaging antibodies:

  • Super-resolution microscopy-optimized antibodies with minimal linkage error

  • Self-labeling antibody tags for live-cell imaging applications

  • Antibody-based biosensors to detect ZDHHC23 conformational changes or activity

• Mass cytometry applications:

  • Metal-conjugated antibodies for CyTOF analysis of ZDHHC23 in heterogeneous cell populations

  • Multiplexed antibody panels to correlate ZDHHC23 with activation state markers

  • Single-cell proteomics approaches using antibody-based capture

• Proteomics-enhanced antibodies:

  • Integration with MS-compatible labeling strategies for enhanced detection sensitivity

  • Development of antibodies specifically validated for immunoprecipitation-mass spectrometry workflows

  • Targeted proteomics approaches using antibody-based enrichment before MS analysis

• Conditional systems:

  • Inducible nanobody expression systems for acute interference with ZDHHC23 function

  • Split-antibody complementation systems to detect ZDHHC23 interactions in living cells

  • Optogenetic antibody systems for spatiotemporally controlled modulation of ZDHHC23

These innovative approaches could address the current challenges in ZDHHC23 detection and characterization, particularly the issues with antibody specificity and sensitivity that have been documented in the literature .