Recombinant Mouse Putative palmitoyltransferase ZDHHC22 (Zdhhc22)

What is the structural composition of mouse ZDHHC22?

Mouse ZDHHC22 is a 263 amino acid multi-pass membrane protein that contains one DHHC-type zinc finger domain. The protein has multiple transmembrane domains consistent with its function as a membrane-bound enzyme. Its amino acid sequence is: MLALRLLNVVAPAYFLCISLVTFVLQLFLFLPSMREDPTATPLFSPAVLHGALFLFLSANALGNYVLVIQNSPDDLGTCQGTMSQRPQCPPPSTHFCRVCSRVTLRHDHHCFFTGNCIGS RNMRNFILFCLYTSLACLYSMVAGVAYISAVLSISFAHPLAFLTLLPTSISQFFSGAVLGSDMFVILMLYLWFAVGLACAGFCCHQLLLILRGQTRYQVRKGMAVRARPWRKNLQEVFGKRWLLGLLVPMFNVGTESSKQQDK . This structural composition is essential for its enzymatic function in protein palmitoylation.

What is the primary catalytic function of ZDHHC22?

ZDHHC22 functions as a palmitoyltransferase that catalyzes the transformation of palmitoyl-CoA and cysteine residues on target proteins to form S-palmitoyl proteins, releasing free CoA in the process . This post-translational modification regulates protein stability and protein-protein interactions . In particular, ZDHHC22 mediates palmitoylation of KCNMA1, regulating its localization to the plasma membrane, and may also mediate palmitoylation of CNN3 . Recent research has identified mTOR as another significant substrate, with ZDHHC22-mediated palmitoylation reducing mTOR stability and thereby influencing downstream signaling pathways .

How does ZDHHC22 function within the broader protein interaction network?

ZDHHC22 operates within a network of functionally related proteins, particularly other palmitoyltransferases. Analysis using STRING protein interaction database shows that ZDHHC22 has strong functional connections with other ZDHHC family members, including ZDHHC23 (interaction score 0.748), ZDHHC11 (0.692), ZDHHC4 (0.690), ZDHHC13 (0.686), and ZDHHC17 (0.668) . These interactions suggest coordination among different palmitoyltransferases in regulating cellular processes. ZDHHC22 also interacts with proteins involved in immune signaling (TRIM59) and other zinc finger proteins, indicating a potential role in diverse cellular pathways beyond direct palmitoylation activities .

What expression systems are optimal for producing recombinant mouse ZDHHC22?

Two primary expression systems have been validated for producing functional recombinant mouse ZDHHC22:

• E. coli expression system: Used for producing His-tagged full-length ZDHHC22 protein (1-263 aa) . While this system is efficient for producing large quantities of protein, it may lack post-translational modifications present in mammalian cells.

• HEK293 expression system: Preferred for applications requiring properly folded and post-translationally modified ZDHHC22, such as pre-coupled magnetic beads formulations . This system better preserves the native conformation and activity of the protein.

The choice between these systems should be guided by the specific research requirements, particularly whether enzymatic activity or structural studies are the primary focus.

What are the recommended storage and handling protocols for recombinant mouse ZDHHC22?

For optimal stability and activity of recombinant mouse ZDHHC22, follow these evidence-based handling protocols:

• Lyophilized protein:

  • Store at -20°C/-80°C upon receipt

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration (50% recommended) for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

• Protein solutions:

  • Store working aliquots at 4°C for up to one week

  • For long-term storage, keep at -20°C/-80°C in buffer containing glycerol

• Pre-coupled magnetic beads:

  • Store at 2-8°C

  • Do not freeze-thaw the beads

  • Stable for at least 6 months under proper storage conditions

Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided for all formulations.

What experimental applications are suitable for recombinant mouse ZDHHC22?

Recombinant mouse ZDHHC22 can be employed in multiple research applications:

How is ZDHHC22 expression altered in breast cancer?

ZDHHC22 expression exhibits a distinct pattern in breast cancer that correlates with tumor subtypes and clinical outcomes:

• Expression patterns: ZDHHC22 expression is significantly lower in estrogen receptor (ER) negative breast cancer tissues and cell lines compared to ER-positive cases . Analysis of The Cancer Genome Atlas (TCGA) data shows that high ZDHHC22 expression is significantly associated with positive estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status .

• Prognostic significance: Higher ZDHHC22 expression is associated with better relapse-free survival in breast cancer patients, suggesting its potential as a prognostic biomarker .

• Epigenetic regulation: The reduced expression of ZDHHC22 in breast cancer appears to be caused by promoter methylation, a common epigenetic silencing mechanism in cancer .

• Subtype correlation: ZDHHC22 expression is decreased in HER2-enriched and basal-like breast carcinoma, which are considered more aggressive subtypes .

These findings establish ZDHHC22 as a potential tumor suppressor with prognostic value in breast cancer.

What is the molecular mechanism by which ZDHHC22 inhibits breast cancer cell proliferation?

ZDHHC22 exerts its anti-proliferative effects in breast cancer through a specific mechanism centered on mTOR regulation:

• mTOR palmitoylation: ZDHHC22 directly palmitoylates mTOR, which reduces mTOR stability .

• Downstream signaling effects: Decreased mTOR stability leads to reduced activation of the AKT signaling pathway, which is critical for cancer cell proliferation, survival, and metabolism .

• Enzymatic activity dependence: The tumor-inhibiting effects are dependent on ZDHHC22's palmitoyltransferase activity, as demonstrated by experiments with the C111A mutation (which affects the catalytic activity) .

• In vivo validation: In tumor xenograft models using BT-549 cells, stable expression of ZDHHC22 significantly reduced tumor growth compared to cells expressing empty vector or mutant ZDHHC22 .

• Cellular effects: ZDHHC22 expression induces cell cycle arrest and apoptosis in breast cancer cells, further contributing to its anti-proliferative effects .

This mechanism represents a novel regulatory pathway in cancer biology and highlights the importance of post-translational modifications in cancer progression.

How does ZDHHC22 influence endocrine therapy response in breast cancer?

ZDHHC22 has emerged as a potential modifier of endocrine therapy response in breast cancer, particularly for tamoxifen resistance:

• Restoration of tamoxifen sensitivity: Ectopic expression of ZDHHC22 can restore sensitivity to tamoxifen therapy in tamoxifen-resistant MCF-7R cells .

• Mechanism of action: Tamoxifen-resistant breast cancer cells frequently exhibit mTOR hyperactivation. By reducing mTOR stability through palmitoylation, ZDHHC22 can potentially reverse this resistance mechanism .

• Clinical implications: This finding suggests that ZDHHC22 may serve as both a prognostic biomarker and therapeutic target for patients with endocrine therapy-resistant breast cancer .

• Therapeutic potential: ZDHHC22-mediated palmitoylation provides a new direction for developing targeted treatments for endocrine therapy-resistant breast cancer .

These insights offer potential strategies for overcoming resistance to endocrine therapies, a significant clinical challenge in breast cancer treatment.

How can the C111A mutation be utilized to study ZDHHC22 function?

The C111A mutation in ZDHHC22, where cysteine at position 111 is replaced with alanine, is a valuable tool for dissecting ZDHHC22 function:

• Functional characterization: The mutation likely affects the DHHC domain critical for palmitoyltransferase activity, providing a catalytically inactive control for experiments .

• Experimental applications:

  • Generate ZDHHC22 and ZDHHC22-Mut expression vectors for transfection studies

  • Create stable cell lines expressing wild-type or mutant ZDHHC22 for long-term studies

  • Use in xenograft models to distinguish effects dependent on palmitoyltransferase activity

• Mechanistic insights: Comparative studies between wild-type and C111A mutant ZDHHC22 can help identify which cellular effects are directly dependent on its enzymatic activity versus potential scaffolding functions .

• Research strategy: A recommended approach is to clone ZDHHC22 full-length gene with or without C111A mutation into a suitable expression vector (like pCMV6-Entry), then transfect into target cell lines using Lipofectamine 2000, followed by G418 selection (concentrations: 200 μg/mL for BT-549, 500 μg/mL for SK-BR-3, and 400 μg/mL for YCC-B1) .

What are effective animal models for studying ZDHHC22 function in cancer?

Based on published research, the following animal model has been validated for studying ZDHHC22 in cancer:

• Tumor xenograft model in nude mice:

  • Cell preparation: BT-549 cells stably transfected with ZDHHC22, ZDHHC22-Mut (C111A), or empty vector

  • Injection method: Subcutaneous injection of 1 × 10^8 cells into the mammary fat pads of female nude mice (aged 4-6 weeks, weighing 18-22 grams)

  • Measurement protocol: Xenograft size measured every 2 days using a Vernier caliper

  • Calculation formula: Tumor volume = 0.5 × length × width^2

  • Study duration: 15 days post-injection

  • Analysis methods: Xenografts can be isolated for measurement, fixed in formalin, embedded in paraffin, and sectioned for immunohistochemistry (IHC) or immunofluorescence studies

This model has successfully demonstrated the anti-tumor effects of ZDHHC22 in vivo, confirming findings from in vitro studies.

What bioinformatic approaches can be used to study ZDHHC22 in cancer datasets?

Several bioinformatic approaches have been successfully applied to analyze ZDHHC22 in cancer:

• Gene Expression Omnibus (GEO) dataset analysis:

  • Access datasets such as GSE65194 and GSE21653, which contain large breast cancer cohorts

  • Classify patients into ZDHHC22-high and ZDHHC22-low groups based on median expression level (probe 229805_at)

  • Perform gene set enrichment analysis (GSEA) using Broad Institute GSEA software 4.0

  • Apply statistical thresholds: normal P-value < 0.05 and false discovery rate < 0.25

• The Cancer Genome Atlas (TCGA) analysis:

  • Analyze ZDHHC22 alterations (mutations, deletions, amplifications) across cancer types

  • Investigate associations between ZDHHC22 expression and clinicopathologic features

  • Correlate expression with molecular subtypes of breast cancer (e.g., HER2-enriched, basal-like)

• Protein interaction network analysis:

  • Use STRING database to identify functional partners of ZDHHC22

  • Analyze interaction scores to prioritize the most significant protein-protein interactions

  • Investigate pathways enriched among ZDHHC22 interacting proteins

These approaches can provide valuable insights into ZDHHC22's role in cancer and identify potential therapeutic opportunities.

What are the emerging applications of ZDHHC22 in cancer biomarker development?

ZDHHC22 shows significant potential as a cancer biomarker based on current evidence:

• Breast cancer prognosis: Higher ZDHHC22 expression correlates with better relapse-free survival in breast cancer patients, suggesting its utility as a prognostic biomarker .

• Endocrine therapy response prediction: ZDHHC22 expression may help predict response to tamoxifen therapy, potentially guiding treatment decisions for hormone receptor-positive breast cancer patients .

• Colon cancer early detection: ZDHHC22 is considered a potential marker for early detection of colon neoplasia, expanding its relevance beyond breast cancer .

• Methylation-based biomarker: The promoter methylation status of ZDHHC22 could serve as an epigenetic biomarker in cancer diagnostics .

Future research should focus on validating these potential applications in larger clinical cohorts and developing standardized assays for clinical implementation.

How might targeted modulation of ZDHHC22 activity be developed as a therapeutic strategy?

Several approaches could be explored to leverage ZDHHC22 in cancer therapy:

• Epigenetic modifiers: Since ZDHHC22 expression appears to be regulated by promoter methylation, DNA methyltransferase inhibitors might restore ZDHHC22 expression in cancers where it is downregulated .

• Palmitoylation-enhancing compounds: Molecules that enhance the palmitoyltransferase activity of ZDHHC22 could potentially amplify its tumor-suppressive effects, particularly in targeting mTOR stability.

• Combination with mTOR inhibitors: Given ZDHHC22's role in regulating mTOR, combination therapies with existing mTOR inhibitors could provide synergistic effects in cancer treatment.

• Endocrine therapy resistance reversal: Development of strategies to upregulate ZDHHC22 specifically in tamoxifen-resistant cells could help restore sensitivity to endocrine therapy .

• Gene therapy approaches: Viral vector-mediated delivery of ZDHHC22 could be explored for tumors with low expression, though significant challenges remain in targeted delivery.

These potential therapeutic strategies highlight the translational relevance of ZDHHC22 research in oncology.