Recombinant Human Cutaneous T-cell lymphoma-associated antigen 9 (CTAGE9)

What is CTAGE9 and what is its structural characterization?

CTAGE9 (cTAGE family member 9) is a human protein encoded in the genome with UniProt accession A4FU28/CTGE9_HUMAN. It belongs to the cTAGE (Cutaneous T-cell lymphoma-associated antigen) family of proteins, which have been implicated in protein trafficking and secretion mechanisms .

To characterize CTAGE9 structure:

• Begin with bioinformatic analysis using databases like UniProt and iPTMnet

• Perform sequence alignments with other CTAGE family members to identify conserved domains

• Consider X-ray crystallography or cryo-EM for structural determination

• Analyze post-translational modification sites that may influence protein folding and function

CTAGE9 undergoes multiple post-translational modifications, suggesting involvement in regulated cellular processes requiring precise protein modification .

What post-translational modifications occur in CTAGE9 and how can they be studied?

CTAGE9 undergoes extensive post-translational modifications that likely regulate its function and interactions. These modifications include:

To study these modifications:

• Use phospho-specific antibodies for detection via western blot or immunoprecipitation

• Employ mass spectrometry-based phosphoproteomics for site identification

• Perform site-directed mutagenesis to create phospho-mimetic or phospho-deficient mutants

• Utilize kinase prediction tools to identify potential regulatory kinases

• Correlate modifications with cellular conditions or stimuli

How is CTAGE9 gene expression regulated in normal tissues?

CTAGE9 expression appears to be context-dependent and tissue-specific. While comprehensive expression data across all tissues is limited, notable findings include:

• A sharp increase in expression specifically during the mid-secretory (MS) phase of the endometrial cycle, suggesting hormone-responsive regulation

• Expression patterns can be analyzed across various cell types according to the Human Protein Atlas data

Methodological approaches to study CTAGE9 expression:

• Quantitative RT-PCR with carefully designed primers that distinguish between CTAGE family members

• RNA-sequencing for transcriptome-wide expression analysis

• In situ hybridization for spatial localization in tissues

• Reporter gene assays to identify promoter and enhancer elements

• ChIP-seq to identify transcription factors regulating CTAGE9 expression

What is the significance of CTAGE9 copy number variation in genomic studies?

CTAGE8/CTAGE9 represents a truly copy number (CN) variable region in the human population, contrary to earlier assumptions of false duplication in reference genomes . This genomic complexity has significant implications for research:

• CN variations may affect gene dosage and subsequent protein expression levels

• Population differences in CN may contribute to disease susceptibility variation

• Reference genome discrepancies can complicate genomic analyses

Methodological approaches:

• Long-read sequencing technologies (e.g., PacBio HiFi) are critical for resolving these complex genomic regions

• Tools like Paraphase enable high-throughput CN detection and genotyping, even at standard whole genome sequencing depth (30X)

• When analyzing paralog groups with high similarity (>99%), a paralog group-centered approach is recommended rather than relying solely on reference genome alignments

• CN analysis should consider population diversity and not just individual genomes

What evidence links CTAGE9 to cancer development or progression?

Several lines of evidence suggest potential roles for CTAGE9 in cancer development:

• PTM site variants have been associated with multiple cancer types:

  • S65L variant observed in skin cancer (DOID:4159)

  • S327C variant in urinary bladder cancer (DOID:11054)

  • T403I variant in prostate cancer (DOID:10283)

  • Y433F variant in skin cancer (DOID:4159)

• These variants affect key phosphorylation sites, suggesting disruption of normal signaling pathways

Methodological approaches for investigating CTAGE9 in cancer:

• Multi-omics analysis integrating genomic, transcriptomic, and proteomic data

• CRISPR-based functional genomics to assess oncogenic potential

• Patient-derived xenograft models to study variant effects in vivo

• Correlation studies between CTAGE9 expression/mutation and clinical outcomes

• Phosphoproteomic analysis of cancer samples to identify altered PTM patterns

What experimental models are available for studying CTAGE9 function?

Several research models and tools are available for CTAGE9 functional studies:

• CTAGE9 knockdown cell lines: Commercially available cell lines engineered with shRNA targeting CTAGE9 delivered via lentiviral vectors. These lines enable loss-of-function studies with knockdown efficiency validated by qRT-PCR .

• Expression systems: Recombinant expression systems for producing CTAGE9 protein, allowing for:

  • Structure-function analyses

  • Protein-protein interaction studies

  • Biochemical characterization

• Animal models: While not explicitly mentioned in the provided references, conditional knockout or transgenic animal models could be developed to study CTAGE9 in vivo.

When selecting experimental models, researchers should consider:

• The need for paralog-specific targeting due to high sequence similarity between CTAGE family members

• Cell type relevance based on endogenous CTAGE9 expression

• The impact of post-translational modifications on function

• Potential compensatory mechanisms from other CTAGE family members

How does CTAGE9 expression change during the endometrial cycle and what are the implications for reproductive biology?

CTAGE9 shows a distinctive expression pattern in the endometrium, with a sharp increase specifically during the mid-secretory (MS) phase of the endometrial cycle . This phase-specific expression suggests potential roles in:

• Endometrial receptivity for embryo implantation

• Decidualization processes

• Hormone-responsive gene regulation

• Endometrial remodeling during the menstrual cycle

Methodological approaches for investigating CTAGE9 in reproductive biology:

• Primary endometrial cell cultures across different cycle phases

• Endometrial organoid models with hormone treatments

• Comparison between normal endometrium and endometriosis samples

• Targeted gene modification in endometrial cell lines

• Correlation with reproductive hormones and other phase-specific markers

This temporal regulation warrants further investigation into CTAGE9's potential roles in both normal reproductive physiology and pathological conditions like endometriosis .

What are the challenges in developing specific detection tools for CTAGE9?

Developing specific detection tools for CTAGE9 presents several challenges:

• Sequence similarity with other CTAGE family members: CTAGE8 and CTAGE9 form a paralog group with high sequence similarity, creating potential cross-reactivity issues .

• Copy number variation: The variable copy number of CTAGE9 across populations complicates quantitative assessments .

• Post-translational modifications: The extensive PTMs on CTAGE9 may affect epitope accessibility and antibody recognition .

Methodological strategies to overcome these challenges:

• Design detection tools targeting unique sequence regions that differentiate CTAGE9 from other family members

• Validate antibody specificity using CTAGE9 knockdown or knockout models

• Develop paralog-specific PCR primers for transcript detection

• Use multiple detection methods for cross-validation

• Consider modification-specific antibodies for studying particular PTM states

How can CTAGE9 function be investigated in relation to endometriosis?

Given the increased expression of CTAGE9 during the mid-secretory phase of the endometrial cycle , investigating its potential role in endometriosis presents an important research direction. Endometriosis is characterized by endometrial-type mucosa outside the uterine cavity with symptoms including painful periods .

Methodological approaches:

• Compare CTAGE9 expression between normal endometrium and endometriotic lesions

• Correlate expression with disease severity and symptom profiles

• Establish in vitro models using CTAGE9 knockdown in endometrial cells

• Investigate the effect of hormone treatments on CTAGE9 expression

• Examine potential interactions with known endometriosis biomarkers

Research design considerations:

• Include appropriate controls matching menstrual cycle phase

• Account for hormonal status and treatments

• Consider multiple endometriotic lesion types and locations

• Integrate with other biomarkers for comprehensive analysis

• Correlate findings with clinical parameters

What are the optimal methods for recombinant CTAGE9 protein production and purification?

For successful recombinant CTAGE9 production:

• Expression system selection:

  • Mammalian expression systems (HEK293, CHO) are preferred for proper post-translational modifications

  • Insect cell systems (Sf9, High Five) for higher yield with some mammalian-like modifications

  • Bacterial systems may be suitable for structural studies of domains lacking critical PTMs

• Vector design considerations:

  • Include appropriate affinity tags (His, FLAG, GST) for purification

  • Consider removable tags via protease cleavage sites

  • Optimize codon usage for the chosen expression system

  • Include secretion signals if needed

• Purification strategy:

  • Multi-step purification combining affinity chromatography with size exclusion and/or ion exchange

  • PTM-specific enrichment methods for studying modified forms

  • Native vs. denaturing conditions based on structural requirements

• Quality control:

  • SDS-PAGE and western blotting for purity and identity

  • Mass spectrometry to verify sequence and modifications

  • Functional assays to confirm biological activity

How can researchers properly validate CTAGE9 knockdown efficiency?

When establishing and validating CTAGE9 knockdown models:

• Transcript level validation:

  • qRT-PCR with primers specific to CTAGE9, avoiding cross-detection of other CTAGE family members

  • RNA-seq for genome-wide expression analysis and specificity confirmation

  • Northern blotting for direct visualization of transcript levels

• Protein level validation:

  • Western blotting with validated antibodies

  • Immunofluorescence for cellular localization

  • Mass spectrometry-based proteomics for quantitative assessment

• Functional validation:

  • Phenotypic assays relevant to hypothesized CTAGE9 function

  • Rescue experiments with recombinant CTAGE9 to confirm specificity

  • Analysis of downstream pathways or interacting partners

The commercially available CTAGE9 knockdown cell lines use optimized shRNA delivered via lentivirus, with knockdown levels determined via qRT-PCR . Researchers should verify knockdown efficiency in their specific experimental context and cell type.

What bioinformatic approaches are recommended for analyzing CTAGE9 in genomic datasets?

Given the complexity of CTAGE9 genomics, specialized bioinformatic approaches are necessary:

• Copy number analysis:

  • Tools like Paraphase specifically designed for paralog groups with high sequence similarity

  • Long-read sequencing data provides better resolution of complex genomic regions

  • Population-level analysis to account for natural variation

• Sequence variant identification:

  • Paralog-aware alignment strategies to avoid misalignment artifacts

  • Careful filtering of variants in high-similarity regions

  • Validation of important variants with alternative methods

• Expression analysis:

  • Pseudogene-aware RNA-seq analysis pipelines

  • Transcript-specific mapping strategies

  • Integration with protein expression data when available

• Functional prediction:

  • PTM site conservation analysis across species

  • Structural modeling to predict variant impacts

  • Interaction network analysis to predict functional pathways

As shown in genome-wide profiling studies, conventional analysis pipelines may misclassify CTAGE8/CTAGE9 as false duplications, highlighting the importance of specialized approaches for segmental duplication regions .

What is the potential role of CTAGE9 in reproductive disorders beyond endometriosis?

Given the significant upregulation of CTAGE9 during the mid-secretory phase of the endometrial cycle , its potential role may extend to multiple reproductive disorders:

• Implantation failure: The mid-secretory phase coincides with the window of implantation, suggesting CTAGE9 may influence endometrial receptivity

• Recurrent pregnancy loss: Dysregulation could affect decidualization and maternal-fetal interface development

• Endometrial cancer: Altered expression patterns may contribute to pathological processes

Research methodology for investigating these connections:

• Compare CTAGE9 expression between normal and pathological samples across different reproductive disorders

• Correlate expression with clinical outcomes in fertility treatment

• Investigate hormonal regulation pathways controlling CTAGE9 expression

• Develop in vitro models mimicking specific reproductive processes with CTAGE9 modulation

• Explore potential biomarker applications in reproductive medicine

How might CTAGE9 variants contribute to cancer progression mechanisms?

The association of CTAGE9 variants with multiple cancer types raises important questions about mechanistic contributions:

• Signal transduction disruption:

  • Variants affecting phosphorylation sites (S65L, S327C, T403I, Y433F) may alter signaling pathways

  • Changed phosphorylation patterns could modify protein-protein interactions or subcellular localization

• Cell-specific effects:

  • Skin cancer-associated variants (S65L, Y433F) may affect keratinocyte-specific functions

  • Urinary bladder cancer variant (S327C) might influence urothelial biology

  • Prostate cancer variant (T403I) could interact with androgen-responsive pathways

Methodological approaches:

• CRISPR-mediated introduction of cancer-associated variants

• Phosphoproteomic comparison between wild-type and variant forms

• Interaction studies to identify disrupted protein complexes

• Cell type-specific functional assays reflecting cancer hallmarks

• In vivo modeling of variant effects on tumor development and progression

What novel therapeutic strategies might target CTAGE9 or its pathways?

Based on current understanding, several therapeutic strategies could be explored:

• Small molecule inhibitors:

  • Target specific PTM enzymes modifying CTAGE9

  • Disrupt protein-protein interactions involving CTAGE9

  • Modulate CTAGE9 expression through transcriptional regulation

• Biologics-based approaches:

  • Antibodies targeting accessible CTAGE9 epitopes

  • Engineered protein domains interfering with CTAGE9 function

  • RNA-based therapeutics for specific knockdown

• Precision medicine applications:

  • Stratification based on CTAGE9 variant status

  • Combined targeting with other pathway components

  • Biomarker development for treatment response prediction

Research methodologies to explore these strategies:

• High-throughput screening for small molecule modulators

• Structure-based drug design targeting CTAGE9 functional domains

• Animal models evaluating candidate therapeutic approaches

• Patient-derived models for personalized therapy testing

• Combination studies with standard-of-care treatments for relevant diseases

What are the most critical knowledge gaps in CTAGE9 research?

Despite emerging data on CTAGE9, several critical knowledge gaps remain:

• Fundamental function: The precise cellular and molecular functions of CTAGE9 remain poorly defined

• Tissue-specific roles: Beyond endometrial expression, comprehensive tissue-specific functions need clarification

• Regulatory mechanisms: Factors controlling CTAGE9 expression, including hormonal regulation, require further investigation

• Interaction network: The protein-protein interaction landscape of CTAGE9 remains largely unexplored

• Paralog-specific functions: Differentiation between CTAGE9 and other family members' functions needs clarification

Addressing these gaps will require complementary research approaches from multiple disciplines, including structural biology, cell biology, genomics, and clinical research.

What multidisciplinary approaches would accelerate CTAGE9 research?

Advancing CTAGE9 research will benefit from integrative approaches combining:

• Structural biology: Determining three-dimensional structure and identifying functional domains

• Systems biology: Mapping interaction networks and pathway integration

• Genomic medicine: Connecting genomic variation with disease phenotypes

• Reproductive biology: Exploring endometrial functions and fertility implications

• Cancer biology: Investigating oncogenic mechanisms and therapeutic applications

• Bioinformatics: Developing specialized tools for analyzing paralogous genes

Collaborative research frameworks combining these approaches would accelerate understanding of CTAGE9 biology and potential clinical applications.