Recombinant Human Peroxisomal membrane protein 4 (PXMP4)

What is the genomic location and structure of human PXMP4?

Human PXMP4 gene is located on chromosome 20 and encodes the peroxisomal membrane protein 4, also known as 24kDa peroxisomal intrinsic membrane protein . The protein is an integral component of the peroxisomal membrane and is expressed across multiple tissue types. The gene structure includes coding regions for both membrane-spanning domains and functional regions involved in peroxisomal processes.

To investigate PXMP4 genomic structure, researchers typically employ:

• Genomic database analysis (NCBI, Ensembl)

• PCR-based mapping of the gene

• Sequencing of cDNA and genomic DNA

• In silico analysis of promoter regions and transcription factor binding sites that regulate PXMP4 expression

How is PXMP4 integrated into the peroxisomal membrane?

PXMP4 integration into peroxisomal membranes involves a complex process requiring the peroxisomal importomer machinery. Studies using deletion mutagenesis have demonstrated that the N-terminal region containing matrix and transmembrane domains is both necessary and sufficient for peroxisomal targeting . The importomer components, including Pex5, Pex13, and Pex14, are crucial for proper PXMP4 integration.

The methodology to study PXMP4 membrane integration includes:

• Protease protection assays to determine protein topology

• Fluorescence microscopy with tagged PXMP4 variants

• Deletion mutagenesis to identify targeting signals

• In vitro reconstitution of membrane insertion using purified components

• Genetic screening in yeast or mammalian cell models using importomer mutants

What are the key experimental models used to study PXMP4 function?

Several experimental models have been developed to investigate PXMP4 function:

• Cell culture models: Human cell lines with PXMP4 overexpression or knockdown

• Mouse models: PXMP4-deficient mice show altered peroxisomal function

• Yeast models: Used for genetic screens of peroxisomal membrane protein integration

• In vitro reconstitution systems: Using purified components to study membrane integration

Each model offers specific advantages:

• Cell lines provide accessibility for biochemical and imaging studies

• Mouse models allow for systemic physiological assessment

• Yeast models facilitate rapid genetic manipulation and screening

• In vitro systems enable precise mechanistic investigations under controlled conditions

How does PXMP4 expression correlate with cancer progression and prognosis?

PXMP4 shows differential expression patterns across cancer types with significant implications for disease progression and patient outcomes. In hepatocellular carcinoma (HCC), PXMP4 mRNA and protein levels are significantly elevated compared to adjacent normal tissues . This overexpression strongly correlates with clinical parameters:

Interestingly, PXMP4 expression patterns vary by cancer type. In prostate cancer, PXMP4 expression is silenced due to intronic CpG dinucleotide-mediated DNA methylation, while in non-small cell lung cancer (NSCLC), PXMP4 expression inversely correlates with CpG island methylation values . PXMP4 has been shown to promote proliferation, invasion, and migration of colorectal cancer cells, suggesting an oncogenic role in this context.

Methodological approaches to study PXMP4 in cancer include:

• Quantitative PCR for mRNA expression analysis

• Western blotting and immunohistochemistry for protein detection

• Methylation-specific PCR and bisulfite sequencing for epigenetic regulation

• Survival analysis using Kaplan-Meier curves and Log-rank tests

• Correlation analysis with established cancer markers (e.g., Ki-67)

What is the role of PXMP4 in the peroxisomal importomer complex?

The peroxisomal importomer is a multi-protein complex responsible for importing both matrix and membrane proteins into peroxisomes. Recent research suggests that the importomer, primarily known for matrix protein import, also functions in the integration of peroxisomal membrane proteins (PMPs) like PXMP4.

The importomer components interact in a coordinated manner:

• PEX5 serves as a receptor for peroxisomal targeting signal 1 (PTS1)-containing proteins

• PEX5 binds to PEX14 with very high affinity in the low nanomolar range

• PEX5 possesses multiple binding sites for PEX14, distributed throughout its N-terminal half

• The N-terminal domain of PEX14 contains a translocation signal that binds PEX5 at a distinct site from matrix proteins

Experimental approaches to elucidate these interactions include:

• Surface plasmon resonance to measure binding affinities

• Recombinant protein expression and purification

• In vitro complex formation and analysis

• Electron microscopy for structural characterization

• Mutagenesis to identify critical interaction domains

How can contradictory findings on PXMP4 function across different tissues be reconciled?

PXMP4 exhibits apparently contradictory roles across different tissues and cancer types, functioning as a tumor promoter in HCC and colorectal cancer while being silenced in prostate cancer. These discrepancies likely reflect tissue-specific functions and regulatory mechanisms.

Approaches to address these contradictions include:

• Tissue-specific conditional knockout models: Generate tissue-specific PXMP4 knockouts to examine function in individual tissues without systemic effects

• Multi-omics integration:

  • Transcriptomics to identify tissue-specific gene expression networks

  • Proteomics to identify tissue-specific binding partners

  • Epigenomics to map methylation patterns across tissues

  • Metabolomics to characterize peroxisomal metabolic profiles

• Context-dependent signaling analysis:

  • Pathway analysis in different cellular contexts

  • Identification of tissue-specific transcription factors

  • Analysis of peroxisome proliferator-activated receptor α (PPARα) regulation across tissues

• Methodological considerations:

  • Standardized expression measurement protocols

  • Validation across multiple cell lines representing each tissue

  • Careful selection of appropriate controls

What experimental approaches are most effective for studying PXMP4 membrane topology?

Understanding PXMP4 membrane topology is crucial for elucidating its function. Several complementary approaches have proven effective:

• Protease protection assays: Differential susceptibility to proteases on either side of the peroxisomal membrane reveals domain orientation . This approach involves:

  • Isolation of intact peroxisomes

  • Treatment with proteases (e.g., proteinase K) in the presence or absence of membrane-disrupting detergents

  • Western blotting with domain-specific antibodies to identify protected fragments

• Fluorescence-based approaches:

  • FRET (Förster Resonance Energy Transfer) analysis of domain proximity

  • Split-GFP complementation to determine domain localization

  • pH-sensitive fluorescent tags to determine lumen vs. cytosolic exposure

• Cysteine accessibility methods:

  • Introduction of cysteine residues at different positions

  • Treatment with membrane-permeable and impermeable thiol-reactive reagents

  • Mass spectrometry analysis of modified sites

• Cryo-electron microscopy:

  • Single-particle analysis of purified PXMP4

  • Tomography of PXMP4 in native membrane environments

How is PXMP4 expression regulated at the transcriptional and epigenetic levels?

PXMP4 expression is subject to complex regulatory mechanisms:

• Transcriptional regulation:

  • Peroxisome proliferator-activated receptor α (PPARα) regulates PXMP4 transcription

  • This suggests modulation by fatty acids and lipid metabolism

• Epigenetic regulation:

  • In prostate cancer, PXMP4 expression is silenced via intronic CpG dinucleotide-mediated DNA methylation

  • In NSCLC, PXMP4 expression inversely correlates with CpG island methylation values

Methodological approaches to study PXMP4 regulation include:

• Reporter gene assays with PXMP4 promoter constructs

• Chromatin immunoprecipitation (ChIP) to identify transcription factor binding

• Bisulfite sequencing to map DNA methylation patterns

• CRISPR-based epigenetic editing to manipulate specific regulatory elements

• RNA stability assays to assess post-transcriptional regulation

What are the functional consequences of PXMP4 deficiency in animal models?

Studies using PXMP4-deficient mice have revealed insights into its physiological roles . Peroxisomes play crucial roles in metabolism of various biomolecules, including lipids and bile acids, and PXMP4 deficiency impacts these processes.

Key experimental approaches include:

• Metabolic phenotyping (glucose tolerance, insulin sensitivity)

• Lipidomic analysis of peroxisomal metabolites

• Histological examination of peroxisome structure and distribution

• Electron microscopy to assess peroxisome morphology

• Functional assays for specific peroxisomal enzymes

• Challenge studies with peroxisome proliferators or high-fat diets

These approaches help distinguish between direct effects of PXMP4 deficiency and compensatory responses, clarifying its role in peroxisomal function and whole-body metabolism.

What are the optimal protocols for purifying recombinant human PXMP4?

Purification of recombinant human PXMP4 presents challenges due to its membrane protein nature. Based on successful approaches with similar peroxisomal membrane proteins, the following protocol is recommended:

• Expression system selection:

  • Bacterial systems: E. coli BL21(DE3) with specialized vectors for membrane proteins

  • Eukaryotic systems: Insect cells (Sf9, High Five) or yeast (P. pastoris) for proper folding

• Construct design:

  • Addition of affinity tags (His6, FLAG, etc.) at either N- or C-terminus

  • Inclusion of cleavable tags for tag removal post-purification

  • Consideration of fusion partners to enhance solubility

• Solubilization optimization:

  • Screening of detergents (DDM, LDAO, DMNG) for efficient extraction

  • Evaluation of solubilization efficiency by Western blotting

  • Optimization of detergent concentration, temperature, and time

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for further purification

• Quality control:

  • SDS-PAGE and Western blotting to confirm purity

  • Mass spectrometry for identity confirmation

  • Circular dichroism to assess secondary structure

  • Dynamic light scattering to evaluate homogeneity

How can researchers effectively develop and validate PXMP4-specific antibodies?

Development of specific antibodies against PXMP4 is crucial for many research applications. A comprehensive approach includes:

• Antigen design strategies:

  • Full-length recombinant protein for polyclonal antibodies

  • Unique peptide sequences (15-25 amino acids) for region-specific antibodies

  • Non-conserved regions to avoid cross-reactivity with other peroxisomal proteins

• Production methods:

  • Monoclonal antibodies: Hybridoma technology or phage display

  • Polyclonal antibodies: Immunization of rabbits or other suitable species

  • Recombinant antibodies: Single-chain variable fragments (scFvs) or nanobodies

• Validation techniques:

  • Western blotting with positive controls (overexpression) and negative controls (knockdown/knockout)

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence with co-localization studies

  • ELISA to determine sensitivity and specificity

  • Cross-adsorption against related proteins to ensure specificity

• Documentation requirements:

  • Detailed protocols for all validation experiments

  • Lot-to-lot variation assessment

  • Determination of optimal working concentrations for different applications

  • Storage conditions and stability data

What are the potential therapeutic implications of targeting PXMP4 in cancer?

Given the differential expression of PXMP4 across cancer types and its correlation with disease progression, PXMP4 presents a potential therapeutic target. Several research directions merit exploration:

• Target validation approaches:

  • CRISPR-Cas9 knockout studies in cancer cell lines and patient-derived xenografts

  • Inducible overexpression systems to assess oncogenic potential

  • In vivo tumor models with PXMP4 modulation

• Therapeutic strategies:

  • Small molecule inhibitors targeting PXMP4 function

  • Monoclonal antibodies against accessible PXMP4 epitopes

  • siRNA or antisense oligonucleotides for expression knockdown

  • Epigenetic modifiers to reverse aberrant PXMP4 expression

• Biomarker development:

  • PXMP4 expression analysis in patient samples for stratification

  • Correlation with response to standard therapies

  • Liquid biopsy approaches for non-invasive monitoring

• Combination approaches:

  • Synergy assessment with standard chemotherapeutics

  • Combination with peroxisome-targeting drugs

  • Integration with immunotherapy approaches

How might PXMP4 function in metabolic disorders related to peroxisomal dysfunction?

Peroxisomes play crucial roles in multiple metabolic pathways, including fatty acid oxidation, bile acid synthesis, and detoxification of reactive oxygen species. As a peroxisomal membrane protein, PXMP4 may contribute to metabolic homeostasis:

• Metabolic phenotyping approaches:

  • Comprehensive metabolomic analysis in PXMP4-deficient models

  • Flux analysis of peroxisomal metabolic pathways

  • Challenge studies with dietary interventions

  • Assessment of peroxisome proliferation in response to metabolic stress

• Clinical investigation directions:

  • PXMP4 expression analysis in metabolic disease tissues

  • Genetic association studies in metabolic disorder cohorts

  • Functional characterization of PXMP4 variants

  • Integration of multi-omics data from patient samples

• Therapeutic considerations:

  • PPARα agonists to modulate PXMP4 expression

  • Metabolic interventions targeting peroxisomal functions

  • Personalized approaches based on PXMP4 expression profiles