Recombinant Human EPM2A-interacting protein 1 (EPM2AIP1)

What is EPM2AIP1 and what are its alternative names in scientific literature?

EPM2AIP1 (EPM2A-interacting protein 1) is a protein that interacts with laforin (EPM2A), and is also known by several alternative names in scientific literature including KIAA0766, My007, and Laforin-interacting protein . The protein plays important roles in various cellular processes and has garnered research interest particularly in relation to its shared promoter with MLH1 and potential applications in cancer research .

What is the amino acid sequence and structural characteristics of human EPM2AIP1?

Human EPM2AIP1 is a 607 amino acid protein with a complex structure. The full amino acid sequence begins with MWMTPKRSKM and continues through defined regions that contribute to its functionality . Recombinant forms of the protein can be expressed as either fragments (such as amino acids 438-579) or as the full-length protein (amino acids 1-607), depending on research requirements . The protein contains multiple functional domains that facilitate its interactions with other cellular components, particularly with EPM2A (laforin).

What is the significance of EPM2AIP1 sharing a promoter with MLH1?

EPM2AIP1 shares a bidirectional promoter with MLH1, which is a critical DNA mismatch repair gene . This shared regulatory region means that factors affecting MLH1 expression, such as promoter hypermethylation, may simultaneously impact EPM2AIP1 expression. This relationship has important implications for cancer research, particularly in colorectal and endometrial cancers where MLH1 promoter hypermethylation is a significant event . The bidirectional nature of this promoter represents an interesting model for studying gene regulation and has prompted investigation into whether EPM2AIP1 expression could serve as a surrogate marker for MLH1 promoter status.

How has EPM2AIP1 immunohistochemistry been evaluated as a surrogate marker for MLH1 promoter hypermethylation?

EPM2AIP1 immunohistochemistry (IHC) has been investigated as a potential surrogate marker for MLH1 promoter hypermethylation (MPH) based on the premise that the shared promoter would lead to concurrent silencing of both genes .

In a key study involving 101 microsatellite instable colorectal cancer cases, researchers systematically evaluated this potential application. The methodology included:

• Selection of cases with known MPH status (74 with MPH, 27 without MPH)

• Performance of EPM2AIP1 IHC on whole tumor sections and tissue microarrays

• Correlation of EPM2AIP1 expression patterns with MLH1 expression and MPH status

• Statistical analysis of sensitivity, specificity, and accuracy

Why was EPM2AIP1 immunohistochemistry found inadequate as a surrogate for MLH1 promoter hypermethylation in colorectal cancer?

EPM2AIP1 immunohistochemistry was ultimately deemed inadequate as a surrogate marker for MLH1 promoter hypermethylation in colorectal cancer for several methodological and biological reasons:

• Low sensitivity and specificity: With only 64% sensitivity and 67% specificity, the test did not meet the reliability standards needed for clinical applications .

• Unexpected staining patterns: A significant number of cases without MLH1 promoter hypermethylation showed loss of EPM2AIP1 expression (33%), indicating that EPM2AIP1 expression is likely regulated by additional mechanisms beyond promoter methylation .

• Inconsistent results in Lynch syndrome cases: Of 10 MLH1-mutated Lynch syndrome cases without promoter hypermethylation, 2 cases unexpectedly showed loss of EPM2AIP1 staining, further undermining the reliability of the marker .

• Variable results in cases with double somatic mutations: Of 6 cases with double somatic mutations of the MLH1 gene without hypermethylation, only 4 demonstrated intact expression of EPM2AIP1 as expected .

Researchers concluded that unless stain quality improves with different antibody clones or platforms, EPM2AIP1 IHC will likely not be useful as a surrogate test for MPH in colorectal cancer .

How does EPM2AIP1's performance as a surrogate marker differ between colorectal and endometrial cancers?

While EPM2AIP1 immunohistochemistry was found inadequate as a surrogate marker in colorectal cancer, previous reports had suggested its utility in endometrial cancer . This discrepancy highlights important tissue-specific differences in gene regulation and protein expression patterns.

Several factors may contribute to this tissue-specific difference:

• Epigenetic landscape variations: The regulatory mechanisms controlling the MLH1/EPM2AIP1 bidirectional promoter may differ between tissue types.

• Tissue-specific transcription factors: Different cofactors may influence promoter activity in endometrial versus colorectal tissues.

• Alternative regulatory mechanisms: Additional tissue-specific mechanisms beyond promoter methylation may influence EPM2AIP1 expression.

• Technical considerations: Differences in tissue fixation, processing, and antibody performance between tissues may affect staining outcomes.

This tissue-specific variation underscores the importance of validating biomarkers in each specific tissue type rather than assuming transferability of results across cancer types.

What expression systems are optimal for producing recombinant human EPM2AIP1 for research purposes?

Based on available information, researchers have successfully produced recombinant human EPM2AIP1 using at least two expression systems, each with distinct advantages depending on research requirements:

• Escherichia coli expression system:

  • Used to produce fragment proteins (e.g., amino acids 438-579)

  • Advantages include high yield and cost-effectiveness

  • Typically results in >50% purity suitable for SDS-PAGE applications

  • May lack post-translational modifications present in mammalian systems

• HEK-293 cells (mammalian expression system):

  • Used to produce full-length proteins (amino acids 1-607)

  • Provides proper folding and post-translational modifications

  • Higher likelihood of producing functionally active protein

  • Purification generally performed via one-step affinity chromatography using His-tag

  • Optimized for intracellular, secreted, and transmembrane proteins

The choice between these systems should be guided by the specific research requirements, including whether post-translational modifications are essential, whether full-length protein is needed, and the intended experimental applications.

How can researchers optimize EPM2AIP1 immunohistochemistry protocols for improved staining quality?

Given the challenges with EPM2AIP1 immunohistochemistry staining quality noted in colorectal cancer research , researchers seeking to improve staining quality might consider the following methodological optimizations:

• Antibody selection and validation:

  • Evaluate multiple antibody clones against EPM2AIP1

  • Perform rigorous validation using positive and negative controls

  • Consider monoclonal antibodies for improved specificity

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize pH conditions for buffer solutions

  • Adjust retrieval duration and temperature

• Signal amplification techniques:

  • Implement polymer-based detection systems

  • Consider tyramide signal amplification for enhanced sensitivity

  • Explore automated staining platforms for consistency

• Tissue preparation considerations:

  • Standardize fixation time to minimize variability

  • Use freshly cut tissue sections when possible

  • Implement strict quality control for tissue processing

• Quantitative analysis approaches:

  • Develop standardized scoring systems

  • Utilize digital image analysis for objective quantification

  • Consider multiplex staining to assess EPM2AIP1 in context with other markers

These technical improvements might enhance the utility of EPM2AIP1 IHC in research contexts, although further validation would be required before clinical applications could be considered.

What purification methods are most effective for isolating recombinant EPM2AIP1 protein?

Effective purification of recombinant EPM2AIP1 typically leverages affinity tags incorporated into the recombinant construct. Based on the available information, the following purification approaches have been utilized:

• His-tag affinity purification:

  • Recombinant EPM2AIP1 is commonly expressed with a histidine tag

  • Purification via one-step affinity chromatography using nickel or cobalt resin

  • This approach allows for relatively straightforward purification with moderate to high purity

• Chromatography optimizations:

  • Buffer composition optimization to maintain protein stability

  • Consideration of additional purification steps for higher purity requirements

  • Potential use of size exclusion chromatography as a polishing step

• Quality assessment:

  • SDS-PAGE analysis to confirm molecular weight and purity

  • Western blotting with anti-EPM2AIP1 antibodies to confirm identity

  • Functional assays to verify biological activity if relevant

When working with EPM2AIP1 fragments (such as amino acids 438-579), researchers have achieved >50% purity suitable for standard applications like SDS-PAGE . For applications requiring higher purity or native conformation, expression in mammalian systems followed by optimized purification protocols may be preferable.

What methodological approaches can be used to study the bidirectional MLH1/EPM2AIP1 promoter regulation?

The bidirectional MLH1/EPM2AIP1 promoter represents an interesting model for studying gene regulation mechanisms. To investigate its regulation, researchers could employ several methodological approaches:

• Chromatin structure analysis:

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding

  • DNase hypersensitivity assays to assess chromatin accessibility

  • ATAC-seq to map open chromatin regions

  • Analysis of nucleosome occupancy at the promoter region

• Methylation studies:

  • Bisulfite sequencing for single-nucleotide resolution of methylation status

  • Methylation-specific PCR for targeted analysis

  • Pyrosequencing for quantitative methylation assessment

  • Correlation of methylation patterns with expression of both genes

• Gene expression analysis:

  • RT-qPCR to quantify transcript levels of both genes

  • RNA-seq for comprehensive transcriptome analysis

  • Single-cell analysis to assess heterogeneity in expression patterns

  • Correlation of expression data with epigenetic modifications

• Functional promoter studies:

  • Reporter assays with promoter constructs driving different fluorescent proteins

  • CRISPR-mediated editing of regulatory elements

  • Analysis of directional transcription using nascent RNA sequencing

  • Investigation of enhancer elements affecting bidirectional expression

These approaches could provide insights into the regulatory mechanisms controlling this bidirectional promoter and potentially identify factors that influence the differential expression patterns observed in different tissues and disease states.

How can researchers investigate the interaction between EPM2AIP1 and laforin (EPM2A)?

To investigate the functional interaction between EPM2AIP1 and laforin (EPM2A), researchers could employ a range of protein-protein interaction and functional analysis methods:

• In vitro interaction assays:

  • Co-immunoprecipitation (Co-IP) using antibodies against either protein

  • GST pull-down assays with recombinant proteins

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Structural analysis approaches:

  • X-ray crystallography of the complex if crystals can be obtained

  • Cryo-electron microscopy for structural determination

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Computational modeling and molecular dynamics simulations

• Cellular localization studies:

  • Immunofluorescence co-localization analysis

  • Fluorescence resonance energy transfer (FRET) for proximal interactions

  • Bimolecular fluorescence complementation (BiFC) in live cells

  • Proximity ligation assay (PLA) for endogenous protein interactions

• Functional assays:

  • Mutational analysis to identify critical interaction domains

  • Phenotypic assays following knockdown/overexpression of either protein

  • Assessment of EPM2AIP1's impact on laforin's phosphatase activity

  • Investigation of pathway alterations when the interaction is disrupted

These methodological approaches would provide complementary insights into the physical and functional relationship between EPM2AIP1 and laforin, potentially revealing the physiological significance of this interaction.

What are the methodological considerations for investigating EPM2AIP1 expression in relation to microsatellite instability in colorectal cancer?

Investigating EPM2AIP1 expression in relation to microsatellite instability (MSI) in colorectal cancer requires careful experimental design and consideration of multiple factors:

• Sample selection and classification:

  • Proper classification of MSI status using standard panels (e.g., Bethesda panel)

  • Inclusion of both MSI-high and microsatellite stable (MSS) cases

  • Stratification based on MLH1 promoter methylation status

  • Consideration of Lynch syndrome cases versus sporadic MSI-high cases

• Comprehensive mismatch repair (MMR) protein analysis:

  • Complete IHC panel including MLH1, PMS2, MSH2, and MSH6

  • Correlation of EPM2AIP1 expression with each MMR protein

  • Analysis of EPM2AIP1 in cases with different MMR deficiency patterns

• Molecular characterization approaches:

  • MLH1 promoter methylation analysis using quantitative methods

  • MLH1 mutation screening in cases without promoter methylation

  • Analysis of double somatic mutations in sporadic cases

  • Integration with other molecular features (BRAF V600E, CIMP status)

• Statistical considerations:

  • Power calculation to determine adequate sample size

  • Multivariate analysis to control for confounding factors

  • Sensitivity, specificity, and accuracy calculations for biomarker evaluation

  • Appropriate statistical tests for categorical and continuous variables

Research has shown that EPM2AIP1 expression patterns in colorectal cancer have complex relationships with MSI status and MLH1 alterations. For example, in a study of 101 MSI cases, EPM2AIP1 loss showed limited sensitivity (64%) and specificity (67%) for MLH1 promoter hypermethylation, with unexpected loss of expression in 33% of cases without hypermethylation . These findings highlight the importance of comprehensive molecular characterization when studying the relationships between these factors.

What is the sensitivity and specificity of EPM2AIP1 immunohistochemistry as a surrogate for MLH1 promoter hypermethylation?

Based on research involving 101 microsatellite instable colorectal cancer cases, the performance metrics of EPM2AIP1 immunohistochemistry as a surrogate marker for MLH1 promoter hypermethylation (MPH) are as follows:

These metrics were derived from the following observations:

• Among 74 cases with confirmed MLH1 promoter hypermethylation, 47 showed loss of EPM2AIP1 expression (true positives), while 27 unexpectedly retained expression (false negatives)

• Among 27 cases without MPH, 18 showed intact EPM2AIP1 expression as expected (true negatives), while 9 showed unexpected loss of expression (false positives)

The relatively low sensitivity and specificity values indicate that EPM2AIP1 immunohistochemistry does not serve as a reliable surrogate marker for MLH1 promoter hypermethylation status in colorectal cancer .

How does EPM2AIP1 expression correlate with different genetic alterations in MLH1?

Research has revealed complex relationships between EPM2AIP1 expression and different genetic alterations affecting MLH1:

These data indicate that while EPM2AIP1 loss is more common in cases with MLH1 promoter hypermethylation, there is substantial inconsistency in this relationship . The unexpected loss of EPM2AIP1 expression in cases without promoter hypermethylation, including Lynch syndrome cases with germline MLH1 mutations, suggests that EPM2AIP1 expression is likely regulated by additional mechanisms beyond the shared promoter with MLH1.

What technical factors might impact the reliability of EPM2AIP1 immunohistochemistry results?

Several technical factors can influence the reliability and reproducibility of EPM2AIP1 immunohistochemistry results, which may partially explain the limitations observed in research studies:

What alternative approaches could be explored for rapid assessment of MLH1 promoter hypermethylation status?

Given the limitations of EPM2AIP1 immunohistochemistry as a surrogate marker, researchers might consider several alternative approaches for assessing MLH1 promoter hypermethylation:

• Direct methylation testing improvements:

  • Development of rapid PCR-based methylation assays

  • Implementation of methylation-sensitive high-resolution melting analysis

  • Exploration of isothermal amplification methods for methylation detection

  • Refinement of pyrosequencing protocols for faster turnaround

• Alternative protein biomarkers:

  • Investigation of other proteins regulated by MLH1 promoter methylation

  • Multiplex IHC panels that combine several markers for improved accuracy

  • Exploration of downstream pathway proteins affected by MLH1 silencing

• Molecular and imaging approaches:

  • Development of methylation-specific probes for in situ hybridization

  • Exploration of metabolic alterations associated with MLH1 silencing

  • Radiomics approaches correlating imaging features with methylation status

• Integrated algorithmic approaches:

  • Machine learning models incorporating multiple biomarkers

  • Risk prediction algorithms that consider clinical and pathological features

  • Integrated molecular-pathological classification systems

Each of these approaches would require rigorous validation before clinical implementation, focusing on metrics such as sensitivity, specificity, cost-effectiveness, and technical feasibility in routine diagnostic settings.

How might further characterization of the EPM2AIP1 protein contribute to understanding its biological functions?

Further characterization of EPM2AIP1 protein could yield valuable insights into its biological functions through several research avenues:

• Structural biology approaches:

  • Determination of three-dimensional structure through X-ray crystallography or cryo-EM

  • Identification of functional domains and active sites

  • Structural comparison with proteins of known function

  • Analysis of post-translational modifications and their impact on function

• Protein interaction studies:

  • Comprehensive interactome mapping using mass spectrometry

  • Yeast two-hybrid screening to identify novel binding partners

  • Investigation of protein complexes involving EPM2AIP1

  • Characterization of the laforin-EPM2AIP1 interaction dynamics

• Functional genomics approaches:

  • CRISPR-Cas9 knockout or knockdown studies

  • Rescue experiments with mutant constructs

  • Tissue-specific conditional knockout models

  • High-throughput phenotypic screening following genetic manipulation

• Physiological role investigations:

  • Examination of tissue-specific expression patterns and regulation

  • Analysis of cellular localization under different conditions

  • Investigation of potential roles in cellular signaling pathways

  • Exploration of disease-relevant functions beyond cancer

These approaches would provide complementary insights into EPM2AIP1's biological roles and potentially reveal new research directions or therapeutic opportunities.

What research questions remain unanswered regarding the bidirectional regulation of the MLH1/EPM2AIP1 promoter?

Several important questions remain unanswered regarding the bidirectional regulation of the MLH1/EPM2AIP1 promoter, representing opportunities for future research:

• Mechanistic questions:

  • What specific transcription factors regulate directional expression from this promoter?

  • How is transcriptional machinery assembled in each direction?

  • What role does chromatin structure play in determining directional bias?

  • Are there tissue-specific regulatory elements that influence directional activity?

• Epigenetic regulation questions:

  • Why does promoter methylation appear to affect MLH1 and EPM2AIP1 expression differently?

  • What other epigenetic marks (histone modifications, etc.) influence bidirectional expression?

  • How stable is the epigenetic regulation of this promoter during development and disease?

  • What triggers promoter hypermethylation in sporadic colorectal cancer?

• Functional relationship questions:

  • Is there a functional relationship between MLH1 and EPM2AIP1 beyond sharing a promoter?

  • Do these proteins participate in common cellular pathways?

  • How did this bidirectional promoter evolve, and is it conserved across species?

  • What is the biological significance of coordinated expression of these genes?

• Clinical and translational questions:

  • Why does EPM2AIP1 IHC appear to have different utility in endometrial versus colorectal cancer?

  • Could combined biomarkers improve the prediction of MLH1 promoter methylation status?

  • Are there therapeutic implications to understanding this bidirectional regulation?

Addressing these questions would advance our understanding of bidirectional promoter regulation in general and potentially provide new insights into the diagnosis and treatment of cancers with MLH1 alterations.