ZDHHC4 Antibody

What is ZDHHC4 and what are its main functions?

ZDHHC4 is a palmitoyltransferase that catalyzes the addition of palmitate onto protein substrates including the D(2) dopamine receptor DRD2, GSK3B, and MAVS. As a member of the ZDHHC family of enzymes, it plays a crucial role in post-translational protein modification through palmitoylation, which can affect protein localization, stability, and function. ZDHHC4 mediates GSK3B palmitoylation to prevent its AKT1-mediated phosphorylation, leading to activation of the STAT3 signaling pathway . It also catalyzes MAVS palmitoylation, which promotes its stabilization and activation by inhibiting 'Lys-48'- but facilitating 'Lys-63'-linked ubiquitination . These activities position ZDHHC4 as an important regulator of various cellular signaling pathways.

The enzyme is also known by several other names including DC1, ZNF374, UNQ5787/PRO19576, Palmitoyltransferase ZDHHC4, Zinc finger DHHC domain-containing protein 4, and Zinc finger protein 374 . Understanding these alternative designations is important when searching scientific literature or databases for information about this protein.

What types of ZDHHC4 antibodies are available for research?

Several types of ZDHHC4 antibodies are available for research applications, with polyclonal antibodies being the most common. These include:

• Rabbit polyclonal antibodies against human ZDHHC4, such as those offered by Atlas Antibodies and Abcam . These antibodies are typically raised against specific regions of the protein.

• Antibodies targeting specific epitopes, such as those corresponding to recombinant fragment proteins within Human ZDHHC4 amino acids 250 to the C-terminus .

• Validated antibodies for specific applications including Western blotting (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC-IF) .

The concentration of commercially available antibodies is typically around 0.1 mg/ml, which is suitable for most research applications . When selecting an antibody, it's important to check the validation data provided by the manufacturer to ensure it's appropriate for your specific experimental needs.

What is the subcellular localization of ZDHHC4?

ZDHHC4 exhibits a distinctive subcellular localization pattern that differentiates it from other ZDHHC family members. The protein is predominantly localized to the endoplasmic reticulum (ER) membranes . This specific targeting is governed by a lysine-based sorting signal, specifically a Lys–Xxx–Xxx (KXX) motif at its C-terminus . This sorting signal is both necessary and sufficient for ER localization, as demonstrated by experiments where adding this targeting signal to typically Golgi-localized ZDHHC enzymes redirects them to the ER.

Structurally, ZDHHC4 has a unique topology with five transmembrane domains (TMDs), whereas most ZDHHC proteins have four TMDs. In ZDHHC4, three TMDs precede the cytoplasmic DHHC-CRD (Asp-His-His-Cys cysteine-rich domain), which is its catalytic domain . This distinctive membrane topology and ER localization likely contribute to ZDHHC4's substrate specificity, as it can only access proteins that transit through or reside in the ER compartment.

What techniques can ZDHHC4 antibodies be used for?

ZDHHC4 antibodies have been validated for multiple research techniques, enabling comprehensive characterization of this protein in various experimental contexts:

• Western Blotting (WB): ZDHHC4 antibodies can be used to detect the protein in cell or tissue lysates, with specific bands corresponding to the expected molecular weight .

• Immunohistochemistry on paraffin-embedded sections (IHC-P): Antibodies have been validated for detection of ZDHHC4 in fixed tissue sections, including human kidney and liver cancer tissues, typically used at dilutions around 1/100 .

• Immunocytochemistry/Immunofluorescence (ICC-IF): These antibodies can visualize the subcellular localization of ZDHHC4 in fixed cells, confirming its ER distribution pattern .

• Immunoprecipitation (IP): ZDHHC4 antibodies can be used to pull down the protein for subsequent analysis, including assessment of its palmitoylation activity or interacting partners .

When using these antibodies, it's important to include appropriate controls and optimize conditions for each specific application to ensure reliable and reproducible results.

How should I design an experiment to study ZDHHC4-mediated protein palmitoylation?

Designing a robust experiment to study ZDHHC4-mediated protein palmitoylation requires careful planning and consideration of multiple variables:

• Define clear objectives:

  • Clearly articulate your hypothesis about ZDHHC4 and its potential substrate

  • Determine whether you're investigating a known interaction or screening for novel substrates

  • Define measurable outcomes that would support or refute your hypothesis

• Select appropriate experimental models:

  • Choose cell lines that express both ZDHHC4 and your protein of interest

  • Consider ZDHHC4 overexpression systems or CRISPR/siRNA knockdown approaches

  • For comparative studies, include other ZDHHC enzymes like GODZ (ZDHHC3)

• Include essential controls:

  • Wild-type ZDHHC4 (positive control)

  • Catalytically inactive ZDHHC4 mutant (negative control)

  • Empty vector transfection (background control)

  • Known ZDHHC4 substrate as positive control (e.g., MOR, GSK3B)

• Select appropriate detection methods:

  • Bioorthogonal click chemistry using 15-hexadecynoic acid (15-HDYA) as a probe

  • Metabolic labeling with radiolabeled palmitate ([3H]-palmitate)

  • Acyl-biotin exchange (ABE) chemistry for non-metabolic detection

• Verify expression and localization:

  • Confirm ZDHHC4 expression by Western blotting

  • Verify substrate expression levels

  • Confirm appropriate subcellular localization (ER for ZDHHC4)

This systematic approach will help ensure that your experiments provide reliable and interpretable data on ZDHHC4-mediated palmitoylation events.

What are the best methods for detecting protein palmitoylation mediated by ZDHHC4?

Several methods can be employed to detect ZDHHC4-mediated protein palmitoylation, each with distinct advantages and limitations:

• Bioorthogonal Click Chemistry (BCC):

  • Uses 15-hexadecynoic acid (15-HDYA) as a chemical probe that can be incorporated into palmitoylated proteins

  • Combined with protein immunoprecipitation using magnetic beads to isolate specific proteins of interest

  • Allows visualization through in-gel fluorescence imaging or Western blotting

  • Provides a rapid, non-isotopic, and efficient method for assaying palmitoylation status

  • Has been successfully used to demonstrate ZDHHC4-mediated palmitoylation of the mu-opioid receptor (MOR)

• Metabolic Labeling with Radiolabeled Palmitate:

  • Traditional approach using [3H]-palmitate incorporation

  • Provides quantitative assessment of palmitoylation rates

  • Requires appropriate radioactive handling facilities and longer exposure times

• Acyl-Biotin Exchange (ABE):

  • Does not require metabolic labeling, making it suitable for tissues or samples where metabolic incorporation is not feasible

  • Involves hydroxylamine treatment to cleave thioester bonds followed by biotinylation

  • Allows detection of existing palmitoylated proteins rather than newly synthesized ones

For optimal results, experimental conditions should be carefully optimized, including incubation times with palmitate analogs, lysis conditions to preserve thioester bonds, and appropriate controls to distinguish specific ZDHHC4-mediated palmitoylation from background or non-specific signals.

How can I validate the specificity of a ZDHHC4 antibody for my research?

Validating the specificity of a ZDHHC4 antibody is crucial for ensuring reliable experimental results. A comprehensive validation approach should include:

• Control samples analysis:

  • Test the antibody on positive controls (tissues or cells known to express ZDHHC4)

  • Use negative controls (ZDHHC4 knockout or knockdown cells)

  • Compare reactivity in different tissues with varying expression levels

• Multi-technique validation:

  • Perform Western blotting to confirm band size (the expected molecular weight)

  • Conduct immunohistochemistry to assess tissue distribution patterns

  • Use immunofluorescence to verify the expected subcellular localization (ER pattern for ZDHHC4)

  • Consider flow cytometry for quantitative assessment of binding specificity

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • This should significantly reduce or eliminate specific signals

  • Serves as a critical control for specificity verification

• Cross-reactivity assessment:

  • Test against closely related ZDHHC family members

  • Particularly important given sequence similarities among ZDHHC proteins

  • Consider using overexpression systems for different ZDHHC proteins

• Application-specific optimization:

  • For WB: Optimize blocking conditions, antibody dilutions, and incubation times

  • For IHC/ICC: Test different fixation methods and antigen retrieval protocols

  • Document optimal conditions for reproducible results

Remember that antibodies validated for one application (e.g., Western blotting) may not work optimally for others (e.g., immunoprecipitation), necessitating validation for each specific application .

What controls should I include when designing flow cytometry experiments with ZDHHC4 antibodies?

When designing flow cytometry experiments with ZDHHC4 antibodies, include these essential controls to ensure reliable and interpretable results:

• Unstained cells control:

  • Cells processed without any antibody

  • Establishes baseline autofluorescence levels

  • Essential for determining negative population boundaries

• Isotype controls:

  • Control antibodies of the same isotype, concentration, and fluorophore

  • Should lack specificity for your target

  • Controls for non-specific binding due to Fc receptors or other interactions

• Single-color controls:

  • Cells stained with each fluorochrome-conjugated antibody separately

  • Required for compensation when using multiple fluorophores

  • Especially important in multiparameter flow cytometry

• Biological controls:

  • ZDHHC4 overexpression cells (positive control)

  • ZDHHC4 knockdown or knockout cells (negative control)

  • Cells known to express varying levels of ZDHHC4

• Titration series:

  • Test series of antibody dilutions to determine optimal concentration

  • Plot signal-to-noise ratio versus antibody concentration

  • Select concentration that provides maximal specific signal with minimal background

• Blocking controls:

  • Test specific blocking of ZDHHC4 epitope with immunizing peptide

  • Include Fc receptor blocking when analyzing cells with potential Fc receptors

• Permeabilization controls:

  • Since ZDHHC4 is an intracellular ER protein, compare different permeabilization methods

  • Balance between adequate permeabilization and preservation of cellular integrity

Proper experimental preparation and design are critical for successful flow cytometry, including knowledge of cellular targets and appropriate antibodies, as well as optimization of sample preparation techniques .

How does ZDHHC4 structure differ from other ZDHHC family members and how might this affect its function?

ZDHHC4 exhibits several structural features that distinguish it from other ZDHHC family members, potentially influencing its substrate specificity and enzymatic function:

• Transmembrane topology:

  • ZDHHC4 contains five transmembrane domains (TMDs), while most ZDHHC proteins have four TMDs

  • Specifically, ZDHHC4 has three TMDs preceding the cytoplasmic DHHC-CRD (cysteine-rich domain), creating a unique orientation

  • This differs from ZDHHC24 (also with five TMDs) where the extra TMD is situated near the C-terminal domain

  • The unique TMD arrangement may position the catalytic DHHC domain differently relative to substrates

• Subcellular targeting mechanisms:

  • ZDHHC4 possesses a specific Lys–Xxx–Xxx (KXX) motif at its C-terminus that directs it to the endoplasmic reticulum

  • This targeting signal differs from the KKXX motif found in ZDHHC6 (also ER-localized)

  • The KXX motif is both necessary and sufficient for ER targeting, as demonstrated by experiments where adding this signal to Golgi-localized enzymes redirects them to the ER

• Functional implications:

  • ER localization restricts ZDHHC4 to palmitoylating proteins that transit through or reside in the ER

  • This spatial segregation from other ZDHHCs (in Golgi or plasma membrane) creates compartmentalized palmitoylation machinery

  • The distinctive membrane topology may create unique substrate binding interfaces or recognition surfaces

These structural differences likely contribute to ZDHHC4's substrate specificity and enzymatic properties, enabling it to palmitoylate specific targets like GSK3B and MAVS with distinct functional outcomes .

What is known about the substrate specificity of ZDHHC4 compared to other ZDHHC enzymes?

ZDHHC4 demonstrates distinctive substrate preferences compared to other ZDHHC enzymes, reflecting its unique structural and localization properties:

• Identified ZDHHC4 substrates:

  • D(2) dopamine receptor (DRD2) - involved in dopaminergic signaling

  • Glycogen synthase kinase-3 beta (GSK3B) - mediates STAT3 pathway activation

  • Mitochondrial antiviral signaling protein (MAVS) - regulates antiviral responses

  • Mu-opioid receptor (MOR) - involved in opioid signaling

• Comparative palmitoylation efficiency:

  • Experimental data shows ZDHHC4 increases MOR palmitoylation by 113% when overexpressed

  • In contrast, GODZ (ZDHHC3) produces a stronger effect, increasing MOR palmitoylation by 246%

  • This suggests differential substrate preferences or catalytic efficiencies among ZDHHC enzymes

• Determinants of substrate specificity:

  • Subcellular compartmentalization: ZDHHC4's ER localization restricts its access to proteins that transit through the ER

  • Structural recognition elements: The unique five-TMD structure may create distinct substrate binding interfaces

  • Sequence motifs: May recognize specific amino acid sequences or structural features in substrate proteins

• Functional consequences:

  • ZDHHC4-mediated GSK3B palmitoylation prevents AKT1-mediated phosphorylation, leading to STAT3 pathway activation

  • ZDHHC4-mediated MAVS palmitoylation promotes its stabilization by inhibiting 'Lys-48'- but facilitating 'Lys-63'-linked ubiquitination

Understanding these substrate preferences is valuable for predicting potential new ZDHHC4 targets and for developing strategies to selectively modulate specific palmitoylation events in research or therapeutic contexts.

How does ZDHHC4 contribute to cellular signaling pathways through protein palmitoylation?

ZDHHC4 influences multiple cellular signaling networks through strategic palmitoylation of key signaling proteins:

• STAT3 signaling pathway:

  • ZDHHC4 palmitoylates GSK3B (glycogen synthase kinase-3 beta)

  • This palmitoylation prevents AKT1-mediated phosphorylation of GSK3B

  • As a result, GSK3B remains active and contributes to STAT3 pathway activation

  • This represents a novel regulatory mechanism where palmitoylation antagonizes phosphorylation

• Antiviral immune signaling:

  • ZDHHC4 palmitoylates MAVS (mitochondrial antiviral signaling protein)

  • This modification promotes MAVS stabilization by altering its ubiquitination pattern

  • Specifically, ZDHHC4-mediated palmitoylation inhibits 'Lys-48'-linked ubiquitination (which targets proteins for degradation)

  • Simultaneously, it facilitates 'Lys-63'-linked ubiquitination (which often promotes signaling activation)

  • This selective modulation of ubiquitination contributes to antiviral immune responses

• Neurotransmitter signaling:

  • ZDHHC4 palmitoylates the D(2) dopamine receptor (DRD2)

  • While the functional consequences need further investigation, palmitoylation typically affects receptor trafficking, stability, and signaling properties

  • ZDHHC4 can also increase palmitoylation of the mu-opioid receptor (MOR), potentially modulating opioid signaling

• Cross-regulation with other post-translational modifications:

  • The GSK3B example demonstrates how ZDHHC4-mediated palmitoylation can antagonize phosphorylation events

  • This reveals a broader regulatory principle where different post-translational modifications compete or cooperate

  • ZDHHC4's ER localization positions it to modify proteins early in their biosynthetic pathway

These diverse signaling roles highlight ZDHHC4 as an important regulator of multiple cellular processes through its palmitoylation activity.

What approaches can be used to identify novel ZDHHC4 substrates?

Identifying novel ZDHHC4 substrates requires sophisticated methodological approaches that can detect palmitoylation in a ZDHHC4-dependent manner:

• Comparative proteomics approaches:

  • Stable isotope labeling with amino acids in cell culture (SILAC) combined with ZDHHC4 overexpression or knockout

  • Acyl-biotin exchange (ABE) or acyl-resin-assisted capture (Acyl-RAC) followed by mass spectrometry

  • Compare palmitoylated proteomes in the presence vs. absence of ZDHHC4 activity

  • Analyze datasets for proteins showing significant changes in palmitoylation status

• Bioorthogonal labeling strategies:

  • Metabolic labeling with 15-hexadecynoic acid (15-HDYA) in cells with modulated ZDHHC4 expression

  • Click chemistry conjugation to capture probes (biotin, fluorophores)

  • Enrichment of labeled proteins followed by proteomic analysis

  • Quantitative comparison between wild-type and catalytically inactive ZDHHC4 conditions

• Proximity-based approaches:

  • BioID or TurboID fusion with ZDHHC4 to identify proximal proteins

  • APEX2-based proximity labeling combined with proteomics

  • These methods identify proteins that come into close proximity with ZDHHC4, which are potential substrates

• Computational prediction and validation:

  • Prediction algorithms based on known ZDHHC4 substrate features

  • Filter candidates by subcellular localization (ER or proteins trafficking through ER)

  • Targeted validation of predicted substrates using site-directed mutagenesis

  • Assessment of palmitoylation using methods like bioorthogonal click chemistry

• Verification strategies:

  • Direct in vitro palmitoylation assays with recombinant ZDHHC4 and candidate substrates

  • Site-specific mutagenesis of putative palmitoylation sites

  • Functional assays to determine the consequences of palmitoylation on substrate activity

These complementary approaches provide a comprehensive strategy for identifying and validating novel ZDHHC4 substrates, expanding our understanding of its biological functions.

Why might I detect multiple bands when using a ZDHHC4 antibody in Western blotting?

Multiple bands in ZDHHC4 Western blots can arise from several biological and technical factors:

• Post-translational modifications:

  • ZDHHC4 itself is subject to palmitoylation, as demonstrated in bioorthogonal click chemistry assays

  • This and other modifications (phosphorylation, ubiquitination) can create bands of different molecular weights

  • These modifications may vary across cell types or experimental conditions

• Protein isoforms:

  • Alternative splicing may generate different ZDHHC4 isoforms

  • These splice variants could appear as distinct bands

  • The antibody's epitope location determines which isoforms will be detected

• Proteolytic processing:

  • ZDHHC4 might undergo partial degradation during sample preparation

  • Adding fresh protease inhibitors to lysis buffers can minimize this issue

  • Different sample preparation methods (direct lysis in SDS buffer vs. detergent extraction) may yield different patterns

• Cross-reactivity:

  • The antibody may detect other ZDHHC family members with sequence similarity

  • Some antibodies might recognize related proteins like ZNF374 (an alternative name for ZDHHC4)

  • Validation using ZDHHC4 knockdown or knockout samples can help identify specific signals

• Technical considerations for membrane proteins:

  • As a multi-pass membrane protein, ZDHHC4 may not fully denature under standard conditions

  • Incomplete denaturation can lead to aggregates or oligomers appearing as higher molecular weight bands

  • Varying denaturation conditions (temperature, time, reducing agents) may help resolve this issue

To determine which band represents specific ZDHHC4 signal, compare patterns across ZDHHC4 overexpression, knockdown, and wild-type samples, focusing on bands that change consistently with ZDHHC4 expression levels.

How can I optimize immunostaining protocols for ZDHHC4 detection in tissue sections?

Optimizing immunostaining protocols for ZDHHC4 detection in tissue sections requires systematic refinement of multiple parameters:

• Tissue fixation and processing:

  • Test different fixatives (4% paraformaldehyde, formalin, Bouin's solution)

  • Optimize fixation time to balance antigen preservation and antibody accessibility

  • For paraffin sections, ensure complete deparaffinization and hydration

  • Consider frozen sections if paraffin processing affects ZDHHC4 antigenicity

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER): Test different buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Optimize retrieval time and temperature (microwave, pressure cooker, water bath)

  • For membrane proteins like ZDHHC4, more extensive retrieval may be necessary

  • Published protocols have successfully used ZDHHC4 antibodies on paraffin-embedded human kidney and liver cancer tissues

• Blocking and permeabilization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Optimize permeabilization for this multi-pass membrane protein (Triton X-100, saponin, digitonin)

  • Include specific steps to block endogenous peroxidase activity if using HRP detection systems

  • Consider dual blocking (protein and serum) for reduced background

• Antibody incubation:

  • Titrate primary antibody concentration (published protocols have used 1/100 dilution)

  • Test different incubation times and temperatures (4°C overnight vs. room temperature)

  • Optimize secondary antibody selection based on detection system

  • Consider signal amplification systems for low-abundance targets

• Controls and validation:

  • Include tissues known to express ZDHHC4 (kidney, liver)

  • Verify expected subcellular localization (ER pattern)

  • Include negative controls (primary antibody omission, isotype control)

  • Consider dual staining with ER markers to confirm localization pattern

Systematic optimization of these parameters will help achieve specific and reproducible ZDHHC4 immunostaining in tissue sections.

What are common pitfalls in ZDHHC4 palmitoylation assays and how can I avoid them?

Several common pitfalls can compromise ZDHHC4 palmitoylation assays, but they can be mitigated with appropriate strategies:

• Low signal-to-noise ratio:

  • Pitfall: High background or weak specific signal makes data interpretation difficult

  • Solution: Optimize click chemistry reaction conditions (copper catalyst concentration, reaction time)

  • Improve immunoprecipitation efficiency using validated antibodies and optimized protocols

  • Increase metabolic labeling time for better incorporation of palmitate analogs

• False negatives in substrate identification:

  • Pitfall: Failing to detect ZDHHC4-dependent palmitoylation of true substrates

  • Solution: Ensure adequate ZDHHC4 expression and enzymatic activity

  • Verify substrate and ZDHHC4 co-localization (both should access the ER compartment)

  • Use multiple palmitoylation detection methods (click chemistry, ABE, metabolic labeling)

  • Consider that some substrates may require specific cellular contexts or co-factors

• Confounding by endogenous palmitoylation machinery:

  • Pitfall: Difficulty distinguishing ZDHHC4-specific effects from other ZDHHCs

  • Solution: Use ZDHHC4 knockout/knockdown cells as baseline controls

  • Include catalytically inactive ZDHHC4 mutants (DHHC to DHHS mutation)

  • Compare with other ZDHHC enzymes (like GODZ) to establish specificity

• Technical issues with membrane protein handling:

  • Pitfall: Poor solubilization or detection of ZDHHC4 and transmembrane substrates

  • Solution: Use appropriate detergents for membrane protein extraction (CHAPS, DDM, Triton X-100)

  • Optimize sample preparation to prevent protein aggregation

  • Adjust SDS-PAGE conditions for membrane proteins (gel percentage, running buffer)

• Inadequate experimental design:

  • Pitfall: Lack of appropriate controls or unclear hypothesis

  • Solution: Clearly define research questions and appropriate controls before beginning

  • Include positive controls (known ZDHHC4 substrates like MOR)

  • Design experiments with quantifiable outcomes for statistical analysis

By anticipating these common issues and implementing preventive strategies, researchers can improve the reliability and interpretability of ZDHHC4 palmitoylation assays.

How should I approach data analysis for palmitoylation assays involving ZDHHC4?

A systematic approach to data analysis is crucial for extracting meaningful insights from ZDHHC4 palmitoylation assays:

• Quantification methods:

  • For gel-based assays: Use densitometry to quantify band intensities

  • Normalize palmitoylation signals to total protein expression

  • For the bioorthogonal click chemistry approach, measure in-gel fluorescence or blot signal intensity

  • Use replicate samples (n ≥ 3) for statistical analysis

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design (t-test, ANOVA)

  • Include p-value calculations to determine significance of differences

  • Consider the biological relevance of statistically significant changes

  • Present data as fold-change relative to appropriate controls

• Controls interpretation:

  • Compare wild-type ZDHHC4 to catalytically inactive mutants

  • Analyze differences between ZDHHC4 and other palmitoyltransferases (e.g., GODZ)

  • Evaluate hydroxylamine sensitivity to confirm thioester bond specificity

  • Assess baseline palmitoylation in the absence of overexpressed ZDHHC4

• Comparative analysis:

  • Examine relative palmitoylation efficiency across different substrates

  • Compare ZDHHC4-mediated palmitoylation to other ZDHHC enzymes

  • Assess changes in palmitoylation under different cellular conditions

  • Create data visualization that effectively communicates key findings

• Integration with functional data:

  • Correlate palmitoylation levels with functional outcomes

  • For GSK3B, examine STAT3 pathway activation

  • For MAVS, assess changes in ubiquitination patterns and stability

  • Connect palmitoylation data to relevant biological processes

• Technical considerations:

  • Account for background signal in all quantifications

  • Consider technical limitations of detection methods

  • Be transparent about sample sizes and replicate variation in reporting

  • Follow field-standard practices for data presentation and statistical analysis

This structured approach to data analysis enhances the rigor and reproducibility of ZDHHC4 palmitoylation studies, facilitating meaningful interpretation of experimental results.