CPA4 Human

What is CPA4 and what is its primary biological function?

Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family that catalyzes the release of carboxy-terminal amino acids from protein substrates. It functions as a secreted protein that plays a role in establishing the tumor microenvironment. The protein is characterized as a monomer with a molecular mass of approximately 47 kDa as predicted, though it appears as 48 kDa when analyzed by SDS-PAGE under reducing conditions . Human CPA4 is derived from the sequence Gly17-Tyr421 and has measurable enzymatic activity in cleaving specific peptide substrates like Ac-Phe-Thiaphe-OH in experimental settings . CPA4 activity contributes to protein processing and regulation of peptide activity, which may explain its role in both normal physiological processes and disease states .

How is CPA4 typically detected and quantified in research settings?

Multiple methodological approaches are employed for detecting and measuring CPA4:

• Protein Detection Methods:

  • SDS-PAGE visualization with Silver Staining or quantitative densitometry using Coomassie Blue Staining (>95% purity standard)

  • Immunohistochemistry for tissue localization and expression level assessment

  • ELISA assays for quantification in serum and other biological fluids

• Activity Measurement:

  • Enzymatic activity assessment through colorimetric assays using specific peptide substrates with indicators such as DTNB (5,5'Dithio-bis (2-nitrobenzoic acid))

  • Standard specific activity measurements (typically >3,500 pmol/min/μg under specified conditions)

• Gene Expression Analysis:

  • RT-PCR for mRNA quantification

  • RNA sequencing for transcriptomic profiling

  • Computational analysis using databases like Oncomine for comparative expression studies

What are the structural characteristics and biochemical properties of human CPA4?

Human CPA4 exhibits several key structural and biochemical properties relevant to research applications:

• Structural Features:

  • Exists as a monomeric protein

  • Complete protein spans from Gly17 to Tyr421 in the amino acid sequence

  • Often expressed with a C-terminal 10-His tag for purification purposes

• Biochemical Properties:

  • Predicted molecular mass of 47 kDa

  • Observed as 48 kDa protein under reducing conditions in SDS-PAGE

  • Possesses metallocarboxypeptidase activity

  • Specific activity exceeds 3,500 pmol/min/μg when measured with appropriate substrates

• Production Considerations:

  • Successfully expressed in mouse myeloma cell line (NS0)

  • Can be supplied as a 0.2 μm filtered solution in Tris and NaCl

  • Requires storage in manual defrost freezers with avoidance of repeated freeze-thaw cycles

How is CPA4 expression altered in human cancers?

Research has demonstrated significant alterations in CPA4 expression across various cancer types:

These alterations suggest CPA4 plays an important role in cancer development and progression, making it a potential biomarker for both diagnostic and prognostic applications.

What mechanisms link CPA4 to cancer progression?

Several molecular mechanisms connect CPA4 to cancer progression:

• Tumor Microenvironment Modification:

  • CPA4 is secreted from cells to catalyze the release of carboxy-terminal amino acids, potentially establishing favorable conditions in the tumor microenvironment

  • This enzymatic activity may influence extracellular matrix composition and cell-cell interactions

• Pathway Involvement:

  • Gene Set Enrichment Analysis (GSEA) has identified several pathways associated with CPA4 expression, including:

    • Formation of the cornified envelope

    • Keratinization processes

    • Immunoregulatory interactions between lymphoid and non-lymphoid cells

    • Antigen processing and presentation

• Immune System Interactions:

  • CPA4 expression negatively correlates with infiltration levels of certain immune cells, particularly NK CD56bright cells

  • This suggests potential immunosuppressive effects that may facilitate tumor immune evasion

• Angiogenesis Connection:

  • CPA4 may participate in processes related to tumor angiogenesis, which plays key roles in tumor invasion and metastasis

  • This connection places CPA4 in the context of factors like Survivin and vascular endothelial growth factor (VEGF) that promote neo-vascularization

What evidence supports CPA4 as a diagnostic and prognostic biomarker?

Multiple lines of evidence establish CPA4's potential as both a diagnostic and prognostic biomarker:

These findings collectively position CPA4 as a promising biomarker that could enhance both cancer detection and outcome prediction, potentially improving clinical decision-making.

What analytical techniques are employed in comprehensive CPA4 functional studies?

Advanced research on CPA4 utilizes sophisticated analytical approaches:

• Multi-omics Analysis:

  • CPA4 methylation level analysis for epigenetic regulation assessment

  • Differential gene expression analysis between high and low CPA4-expressing samples

  • Gene Set Enrichment Analysis (GSEA) to identify enriched biological pathways

  • Protein-protein interaction mapping to elucidate functional networks

• Immune Contextualization:

  • Tumor Immune Estimation Resource (TIMER2) analysis

  • Single-sample Gene Set Enrichment Analysis (ssGSEA) for immune infiltration correlation

  • Analysis of 24 immune cell types including pDC, NK CD56bright cells, DC, cytotoxic cells, and various T cell populations

• Integrative Approaches:

  • Coexpression analysis to identify genes functionally related to CPA4

  • Development of prognostic models combining CPA4 with other molecular factors

  • Gene Expression Profiling Interactive Analysis using platforms like GEPIA2

These comprehensive methodologies provide multidimensional insights into CPA4's functional role in both normal physiology and disease states.

What are key considerations for designing CPA4 knockdown or overexpression experiments?

When designing genetic manipulation experiments involving CPA4, researchers should consider:

• Model Selection:

  • Cell line choice based on endogenous CPA4 expression and cancer type relevance

  • Consideration of in vitro versus in vivo approaches based on specific research questions

  • Selection of appropriate controls that account for both technical and biological variability

• Manipulation Strategies:

  • For knockdown: siRNA, shRNA, or CRISPR-Cas9 approaches with careful target sequence selection

  • For overexpression: vector selection (transient vs. stable), promoter choice, and tag placement that preserves enzymatic activity

  • Validation at both mRNA and protein levels, with functional assessment of enzymatic activity

• Functional Readouts:

  • Cell proliferation, migration, and invasion assays for cancer-related phenotypes

  • Analysis of CPA4-associated pathways identified through GSEA studies

  • Assessment of immune cell interactions when investigating microenvironment effects

  • Gene expression profiling to identify downstream molecular changes

• Experimental Design Principles:

  • Implementation of research methods following established human development research guidelines

  • Appropriate statistical design with adequate controls and replication

  • Consideration of both acute and long-term effects based on protein stability and turnover

Following these methodological considerations ensures robust and reproducible results in functional studies of CPA4.

How are recombinant CPA4 proteins prepared for experimental applications?

Production of high-quality recombinant CPA4 protein involves several critical steps:

• Expression Systems:

  • Mouse myeloma cell line (NS0) has been successfully employed for expression of human CPA4

  • Expression typically covers the sequence from Gly17 to Tyr421 of the protein

• Design Considerations:

  • Addition of a C-terminal 10-His tag for purification purposes

  • Confirmation of N-terminal sequence (Gly17) through sequence analysis

• Quality Control Measures:

  • Purity assessment >95% by SDS-PAGE with Silver Staining and quantitative densitometry

  • Endotoxin level determination (<1.0 EU per 1 μg of protein by LAL method)

  • Molecular mass verification (predicted 47 kDa; observed 48 kDa in SDS-PAGE)

• Activity Validation:

  • Functional assessment through cleavage of colorimetric peptide substrates

  • Establishment of specific activity levels (>3,500 pmol/min/μg under specified conditions)

• Storage and Handling:

  • Formulation as a 0.2 μm filtered solution in Tris and NaCl

  • Proper shipping conditions with polar packs

  • Storage recommendations including avoidance of repeated freeze-thaw cycles

These standardized approaches ensure consistent production of functional recombinant CPA4 suitable for diverse experimental applications.

What statistical approaches validate CPA4 as a cancer biomarker?

Robust statistical methodologies are employed to establish CPA4's biomarker utility:

These statistical approaches collectively establish the significance of CPA4 alterations in cancer and validate its potential as both a diagnostic and prognostic biomarker.

What potential therapeutic strategies might target CPA4 or its pathways?

Based on current understanding of CPA4 biology, several therapeutic approaches emerge:

• Direct CPA4 Targeting:

  • Small molecule inhibitors of CPA4's enzymatic activity

  • Neutralizing antibodies against CPA4 to prevent its effects in the tumor microenvironment

• Pathway-Based Interventions:

  • Targeting pathways identified through GSEA as associated with CPA4, including:

    • Immunoregulatory pathways between lymphoid and non-lymphoid cells

    • Antigen processing and presentation mechanisms

    • Collagen fibril assembly processes

• Immune-Based Strategies:

  • Approaches to counteract CPA4's negative correlation with certain immune cell populations, particularly NK CD56bright cells

  • Combination with immunotherapies to enhance anti-tumor immune responses

• Expression Modulation:

  • Epigenetic approaches targeting CPA4 methylation patterns

  • RNA interference strategies to reduce CPA4 expression in tumors

While these approaches represent promising directions, they require further validation through preclinical and clinical studies before implementation in cancer treatment regimens.

What are current limitations in CPA4 research methodology?

Despite significant progress, several methodological challenges persist in CPA4 research:

• Mechanistic Understanding:

  • Limited knowledge of specific substrates and molecular mechanisms through which CPA4 influences tumor progression

  • Incomplete understanding of interactions between CPA4 and other components of the tumor microenvironment

• Model Systems:

  • Need for improved in vitro and in vivo models that better recapitulate CPA4's role in human cancers

  • Standardization of systems for studying CPA4 function across different cancer types

• Clinical Translation:

  • Requirement for larger, prospective clinical studies to validate CPA4's diagnostic and prognostic value

  • Standardization of measurement methods for clinical application

  • Establishment of universal cutoff values for diagnostic and prognostic decisions

• Technical Challenges:

  • Optimization of detection methods with improved sensitivity and specificity

  • Development of standardized protocols for measuring CPA4 enzymatic activity in complex biological samples

Addressing these limitations will advance understanding of CPA4 biology and accelerate its translation into clinical applications.

How might emerging technologies enhance CPA4 research?

Several technological advances hold promise for revolutionizing CPA4 research:

• Single-Cell and Spatial Technologies:

  • Single-cell RNA sequencing to understand cell-type-specific expression and effects of CPA4

  • Spatial transcriptomics and proteomics to map CPA4 expression and activity within the tumor microenvironment

• Advanced Proteomics:

  • Improved mass spectrometry techniques for identifying CPA4 substrates and interaction partners

  • Proteomics approaches to map post-translational modifications affecting CPA4 activity

• CRISPR-Based Technologies:

  • CRISPR screening to identify synthetic lethal interactions with CPA4

  • CRISPR base and prime editing for precise manipulation of CPA4 expression and function

• Improved Biomarker Detection:

  • Development of more sensitive methods for detecting CPA4 in liquid biopsies

  • Integration with other biomarkers in multi-analyte panels

  • Longitudinal monitoring approaches using minimally invasive sampling

These technological advances will likely accelerate discovery in CPA4 research and facilitate its translation into clinical applications.

What interdisciplinary approaches might advance CPA4 cancer research?

Integrative research strategies combining multiple disciplines could significantly enhance CPA4 research:

• Computational Biology Integration:

  • Machine learning approaches for integrating CPA4 with other biomarkers

  • Network analysis to understand CPA4 in broader biological systems

  • Structural biology predictions to design specific inhibitors

• Immunology-Oncology Interface:

  • Deeper investigation of CPA4's role in immune evasion mechanisms

  • Exploration of potential synergies between CPA4 inhibition and immunotherapy

  • Assessment of CPA4's influence on tumor-infiltrating immune populations

• Clinical-Basic Science Collaborations:

  • Translational studies linking laboratory findings to patient outcomes

  • Patient-derived models for personalized assessment of CPA4 function

  • Prospective clinical validation of CPA4-based diagnostic and prognostic tools

• Multi-omics Integration:

  • Combined analysis of genomic, transcriptomic, proteomic, and metabolomic data

  • Integration of epigenetic regulation with functional consequences

  • Comprehensive profiling of CPA4-associated molecular signatures across cancer types

These interdisciplinary approaches would provide holistic understanding of CPA4's role in cancer biology and accelerate development of CPA4-based clinical applications.