Recombinant Bovine Peroxisomal membrane protein 4 (PXMP4)

What is Peroxisomal Membrane Protein 4 (PXMP4) and what are its key characteristics?

PXMP4 is a ubiquitously expressed peroxisomal membrane protein with a molecular weight of 24kDa . The gene encoding PXMP4 is located at 20q11.22 in humans and is conserved across various animals including chimpanzees, dogs, cattle, and mice . As an integral membrane protein associated with peroxisomes, PXMP4 is one of the main components of the peroxisome membrane . Its primary known binding partner is ex19, an intracellular chaperone that functions as part of the peroxisome membrane insertion machinery . PXMP4 is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), suggesting its expression may be modulated in response to metabolic changes .

How does bovine PXMP4 compare structurally and functionally to PXMP4 in other species?

Bovine PXMP4 shares significant structural and functional homology with PXMP4 from other species, as the protein is well-conserved across mammals . While the specific differences between bovine PXMP4 and other species are not extensively detailed in current literature, research using recombinant forms demonstrates similar biochemical properties across species . The conservation of PXMP4 across different animals (chimpanzees, dogs, cattle, and mice) suggests it performs similar cellular functions across species . Functional studies in various mammalian systems indicate that PXMP4 is generally involved in peroxisomal membrane maintenance and potentially in specific metabolic pathways, including ether lipid metabolism as suggested by studies in knockout mice .

What expression systems are most effective for producing recombinant bovine PXMP4?

Multiple expression systems have been successfully employed to produce recombinant bovine PXMP4, each with distinct advantages depending on research requirements . The most common expression systems include:

For most research applications requiring high purity (≥85%), all these systems can produce suitable bovine PXMP4 as determined by SDS-PAGE analysis .

What are the optimal methods for detecting and quantifying PXMP4 expression in experimental samples?

Several robust methods have been established for detecting and quantifying PXMP4 expression in experimental samples:

• Quantitative real-time PCR (qPCR): For PXMP4 mRNA detection, primers specific to PXMP4 and a housekeeping gene (like β-Actin) are used with a thermal cycling program featuring an annealing temperature of 60°C . Expression levels are calculated using the 2^-ΔΔCt method, comparing the target gene against the reference gene .

• Western blot analysis: For protein detection, samples are lysed with RIPA buffer, quantified using BCA protein assay, and separated on 12% SDS-polyacrylamide gels . After transfer to PVDF membranes, PXMP4 is detected using specific antibodies (typically at 1:1000 dilution) . Signal quantification is performed using imaging software (e.g., Image J) to analyze band intensity relative to housekeeping proteins like GAPDH .

• Immunohistochemistry: This method allows visualization of PXMP4 expression in tissue sections . Typically employing a 1:200 dilution of PXMP4 rabbit polyclonal antibody, followed by DAB staining and counterstaining . Positive staining appears as brownish-yellow granules in the nucleus, and quantification involves scoring 200 cells under high-power fields (400×) .

Each method has specific advantages: qPCR provides high sensitivity for transcriptional analysis, Western blotting enables protein quantification, and immunohistochemistry reveals spatial distribution within tissues .

How can researchers effectively purify recombinant bovine PXMP4 while maintaining its structural integrity?

Purification of recombinant bovine PXMP4 requires careful consideration of its membrane protein nature to maintain structural integrity:

• Expression system selection: While E. coli systems offer high yields, mammalian or insect cell expression systems better preserve natural folding and post-translational modifications crucial for structural integrity .

• Purification protocol:

  • Initial extraction using gentle detergents (e.g., n-dodecyl β-D-maltoside or digitonin) to solubilize membrane proteins without denaturing them

  • Affinity chromatography utilizing tags (His, GST) incorporated during recombinant expression

  • Size exclusion chromatography to enhance purity and remove aggregates

  • Ion exchange chromatography for final polishing

• Quality assessment: SDS-PAGE analysis should confirm ≥85% purity as the standard benchmark for recombinant bovine PXMP4 . Western blotting with specific antibodies confirms identity and integrity.

• Storage conditions: Purified PXMP4 should be stored in buffer containing appropriate detergent concentrations above their critical micelle concentration, with glycerol (10-20%) and stored at -80°C to prevent aggregation and maintain integrity.

Throughout purification, monitoring protein folding using circular dichroism spectroscopy helps ensure structural integrity is maintained during the purification process.

What validated antibodies and detection reagents are recommended for bovine PXMP4 research?

Several validated antibodies and reagents have been employed successfully in PXMP4 research:

• Primary antibodies:

  • PXMP4 rabbit polyclonal antibody (Novus Biologicals) has been validated for Western blotting (1:1000 dilution) and immunohistochemistry (1:200 dilution)

  • Anti-PXMP4 monoclonal antibodies for specific research applications requiring higher specificity

• Secondary antibodies:

  • Goat anti-rabbit secondary antibodies (1:2000 dilution) have shown good results for detection systems

  • HRP-conjugated or fluorophore-conjugated secondary antibodies depending on detection method

• Detection systems:

  • ECL (enhanced chemiluminescence) development kits for Western blot visualization

  • DAB (3,3'-diaminobenzidine) systems for immunohistochemical applications

• PCR reagents:

  • Specific primer pairs for bovine PXMP4 detection in qPCR applications

  • UltraSYBR Mixture for reliable quantification

When selecting detection reagents, researchers should verify cross-reactivity with bovine proteins specifically, as antibodies raised against human or rodent PXMP4 may have varying degrees of reactivity with bovine PXMP4.

How does PXMP4 contribute to peroxisomal function and lipid metabolism?

PXMP4's contribution to peroxisomal function and lipid metabolism reveals complex interactions:

• Peroxisomal membrane maintenance: As an integral membrane protein, PXMP4 contributes to the structural integrity and functional capacity of peroxisomal membranes .

• Lipid metabolism influence: Studies with PXMP4 knockout mice revealed specific alterations in lipid profiles, particularly:

  • Decreased hepatic levels of alkyldiacylglycerol class of neutral ether lipids

  • Specific reduction in polyunsaturated fatty acid-containing ether lipids

• Phytol metabolism: PXMP4 knockout mice showed elevated levels of phytanic and pristanic acid, suggesting an involvement in peroxisomal α-oxidation pathways responsible for processing these phytol metabolites .

• Interaction with fatty acid processing: While PXMP4 knockout did not significantly alter very long-chain fatty acid (VLCFA) or bile acid levels under standard conditions, the protein likely participates in specialized aspects of peroxisomal lipid metabolism .

• Transcriptional regulation: PXMP4 expression is regulated by PPARα, a key transcription factor controlling lipid metabolism genes, suggesting PXMP4's role may be modulated in response to metabolic demands .

Despite these observations, knockout mice remained viable and fertile with no overt morphological changes to peroxisomes, indicating PXMP4 is not essential for basic peroxisomal function but may be involved in specialized metabolic pathways .

What is the relationship between PXMP4 expression and cancer development?

Research has uncovered significant associations between PXMP4 expression and various cancer types:

• Hepatocellular carcinoma (HCC):

  • PXMP4 mRNA and protein expression are significantly upregulated in HCC tissues compared to adjacent normal tissues

  • High PXMP4 expression correlates with lower differentiation grade, lymph node metastasis, greater invasion depth, and advanced TNM stage

  • Patients with high PXMP4 expression demonstrate poorer survival outcomes

  • The expression rate of PXMP4 was significantly higher in male HCC patients (77.4%) compared to female patients (42%)

• Prostate cancer:

  • PXMP4 expression is silenced primarily through intronic CpG dinucleotide-mediated DNA methylation

  • This presents a contrasting pattern to HCC, suggesting tissue-specific roles

• Non-small cell lung cancer (NSCLC):

  • PXMP4 expression inversely correlates with CpG island methylation values

  • PXMP4 negatively correlates with the proliferation marker Ki-67 protein

• Colorectal cancer:

  • PXMP4 promotes proliferation, invasion, and migration of colorectal cancer cells

  • Expression levels are elevated compared to control tissues

These findings indicate that PXMP4 may serve as a potential biomarker for cancer diagnosis and prognosis, with its expression pattern and function appearing to be cancer-type specific . The mechanisms underlying these associations likely involve PXMP4's role in peroxisomal metabolism and potentially in cellular signaling pathways related to proliferation and metastasis.

What phenotypic changes are observed in PXMP4 knockout models?

Studies using PXMP4 knockout mice have revealed subtle but important phenotypic changes:

The relatively mild phenotype suggests PXMP4 may have specialized functions that become apparent only under specific conditions or may involve subtle metabolic roles that other peroxisomal proteins can partially compensate for in knockout models .

How should researchers analyze PXMP4 expression data in relation to clinical outcomes?

Analysis of PXMP4 expression data in clinical contexts requires rigorous statistical approaches:

• Statistical methods for comparative analysis:

  • For comparing PXMP4 expression between tumor and normal tissues, paired t-tests or Wilcoxon signed-rank tests should be employed

  • Expression differences across multiple groups (e.g., different tumor grades) require ANOVA or Kruskal-Wallis tests

• Correlation with clinicopathological features:

  • Chi-square tests can evaluate associations between PXMP4 expression and categorical variables (gender, tumor differentiation, lymph node metastasis)

  • PXMP4 expression significantly correlates with male gender (77.4% vs. 42% in females), poor differentiation (100% vs. 53.8% in well-differentiated tumors), and lymph node metastasis (92.3% vs. 54% without metastasis)

• Survival analysis approaches:

  • Kaplan-Meier curves with Log-rank tests to assess differences in survival between high and low PXMP4 expression groups

  • Cox proportional hazards models for multivariate analysis to determine independent prognostic value

  • Censoring protocols should clearly define uncensored data (patients who died due to disease) versus censored data (patients still alive at follow-up end or who died from other causes)

• Data presentation standards:

  • Continuous data should be presented as means with standard deviations (x̄ ± s)

  • Immunohistochemical results should be semi-quantitatively scored based on staining intensity and percentage of positive cells

Researchers should consider adjusting for confounding variables and validate findings through independent cohorts when establishing PXMP4 as a prognostic or diagnostic biomarker.

What are the challenges in interpreting PXMP4 functional studies across different model systems?

Interpreting PXMP4 functional studies across different model systems presents several challenges:

• Species-specific variations:

  • While PXMP4 is conserved across mammals, subtle differences in regulation and function may exist between species

  • Recombinant bovine PXMP4 studies may not directly translate to human applications without validation

• Expression system limitations:

  • Different expression systems (E. coli, yeast, baculovirus, mammalian cells, cell-free) produce recombinant PXMP4 with varying post-translational modifications

  • The choice of expression system may influence protein folding, activity, and interaction partners

• Contextual differences in expression patterns:

  • PXMP4 shows tissue-specific expression patterns and disease associations

  • Upregulated in hepatocellular carcinoma but silenced in prostate cancer

  • These opposing patterns highlight the importance of cellular context

• Methodological inconsistencies:

  • Variation in detection methods (qPCR, Western blot, immunohistochemistry) and quantification approaches

  • Antibody specificity differences between studies may affect result interpretation

• Knockout model insights and limitations:

  • PXMP4 knockout mice show subtle phenotypes, suggesting compensatory mechanisms or redundant functions

  • Acute knockdown models may reveal different phenotypes than germline knockouts due to adaptive mechanisms

Researchers should approach cross-model comparisons with caution, validating key findings across multiple systems and confirming relevance to their specific research context.

How do post-translational modifications affect PXMP4 function and how can these be analyzed?

Post-translational modifications (PTMs) of PXMP4 represent an important but understudied aspect of its biology:

• Potential PTMs affecting PXMP4:

  • Phosphorylation sites may regulate membrane localization or protein-protein interactions

  • Ubiquitination could control protein turnover and stability

  • Glycosylation might influence protein folding and trafficking

• Analytical approaches for PTM detection:

  • Mass spectrometry-based proteomics: Ideal for comprehensive PTM mapping

    • Sample preparation should preserve modifications of interest

    • Enrichment strategies (e.g., phosphopeptide enrichment) increase detection sensitivity

    • Fragmentation techniques like electron transfer dissociation better preserve labile modifications

  • Antibody-based methods: For targeted PTM detection

    • Western blotting with modification-specific antibodies

    • Immunoprecipitation followed by PTM-specific detection

  • Site-directed mutagenesis: For functional validation

    • Mutation of potential modification sites can confirm their functional significance

    • Expression of mutant constructs in cellular models to assess phenotypic changes

• Expression system considerations:

  • Mammalian expression systems provide the most physiologically relevant PTM patterns for bovine PXMP4

  • Bacterial systems lack many eukaryotic PTM capabilities and should be avoided when studying modifications

  • Cell-free systems allow incorporation of modified amino acids for studying specific PTM effects

• Functional correlation strategies:

  • Temporal correlation of PTM status with peroxisomal function or metabolic states

  • Modification changes in response to peroxisome proliferators or metabolic stress

  • Comparison of PTM patterns between normal and disease states (e.g., cancer vs. normal tissue)

When analyzing PXMP4 PTMs, researchers should consider that modifications may vary between recombinant and native proteins, necessitating validation in physiologically relevant systems.

What are the key unresolved questions regarding PXMP4's molecular function?

Several critical aspects of PXMP4 function remain unresolved:

• Precise molecular function:

  • Despite identification as a peroxisomal membrane protein, PXMP4's exact biochemical role remains unclear

  • Whether it functions as a channel, transporter, or structural component is not definitively established

  • Its role in specific metabolic pathways requires further delineation

• Binding partners and interaction network:

  • Only ex19 (an intracellular chaperone involved in peroxisome membrane insertion) is currently identified as a binding partner

  • The complete PXMP4 interactome remains unexplored

  • How these interactions change in disease states is unknown

• Regulatory mechanisms:

  • While PPARα transcriptionally regulates PXMP4 , other transcription factors controlling its expression are not well-characterized

  • Post-transcriptional regulation through microRNAs or RNA-binding proteins is unexplored

  • Post-translational modifications affecting PXMP4 function remain uncharacterized

• Role in ether lipid metabolism:

  • The mechanistic basis for decreased alkyldiacylglycerol levels in PXMP4 knockout mice requires investigation

  • Whether PXMP4 directly participates in ether lipid synthesis or transport is unknown

• Tissue-specific functions:

  • Why PXMP4 has opposing roles in different cancer types (upregulated in HCC, silenced in prostate cancer) requires explanation

  • The physiological basis for its higher expression in male versus female HCC patients (77.4% vs. 42%) is not understood

Addressing these questions will require multidisciplinary approaches combining structural biology, metabolomics, and advanced imaging techniques.

How might PXMP4 be exploited as a therapeutic target for cancer and metabolic disorders?

The potential of PXMP4 as a therapeutic target presents several promising avenues:

• Cancer therapy applications:

  • Hepatocellular carcinoma: Inhibition of PXMP4 represents a potential molecular target for HCC treatment given its high expression and correlation with poor prognosis

  • Targeted approaches:

    • Small molecule inhibitors targeting PXMP4 protein function

    • siRNA or antisense oligonucleotides to downregulate PXMP4 expression

    • CRISPR-based gene editing to modify PXMP4 in tumor tissues

• Biomarker development:

  • PXMP4 expression correlates significantly with clinicopathological features including tumor differentiation, lymph node metastasis, and invasion depth

  • Potential applications as:

    • Diagnostic marker for early cancer detection

    • Prognostic indicator for patient stratification

    • Predictive biomarker for treatment response

• Metabolic disorder interventions:

  • PXMP4's involvement in ether lipid metabolism suggests potential applications in disorders involving peroxisomal dysfunction

  • Modulation of PXMP4 might affect phytanic/pristanic acid metabolism, relevant to Refsum disease and other peroxisomal disorders

• Combination therapy strategies:

  • PXMP4 inhibition could potentially sensitize cancer cells to conventional chemotherapy

  • Synergistic approaches targeting PXMP4 alongside other peroxisomal proteins

• Delivery challenges and solutions:

  • As a membrane protein, PXMP4 presents unique targeting challenges

  • Nanoparticle-based delivery systems or peroxisome-targeted compounds might improve specificity

  • Tissue-specific delivery systems would be crucial to target PXMP4 in specific contexts

Development of PXMP4-based therapeutics would require careful consideration of its differential expression patterns across tissues and disease states to maximize efficacy while minimizing side effects .

What novel experimental approaches could advance our understanding of PXMP4 biology?

Innovative experimental approaches could significantly advance PXMP4 research:

• Advanced structural biology techniques:

  • Cryo-electron microscopy to determine high-resolution structures of membrane-embedded PXMP4

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics and interaction surfaces

  • In silico molecular dynamics simulations to predict functional domains and binding pockets

• Single-cell analysis approaches:

  • Single-cell transcriptomics to identify cell populations with differential PXMP4 expression

  • Spatial transcriptomics to map PXMP4 expression patterns within tissues

  • Single-cell proteomics to correlate PXMP4 protein levels with cellular phenotypes

• Advanced genetic models:

  • Tissue-specific and inducible PXMP4 knockout systems to overcome potential developmental compensation

  • Humanized mouse models expressing human PXMP4 variants

  • CRISPR-based screens to identify genetic interactions and synthetic lethalities

• Metabolic flux analysis:

  • Stable isotope labeling to track metabolic pathways affected by PXMP4 manipulation

  • Dynamic metabolomics to determine temporal effects of PXMP4 on peroxisomal metabolism

  • Integration with computational modeling to predict metabolic consequences

• Multi-omics integration:

  • Combined transcriptomic, proteomic, and metabolomic analyses of PXMP4-manipulated systems

  • Network biology approaches to position PXMP4 within cellular pathways

  • Machine learning algorithms to identify patterns in multi-dimensional datasets

• Organoid and patient-derived models:

  • Development of liver organoids to study PXMP4 in a physiologically relevant system

  • Patient-derived xenografts to evaluate PXMP4 targeting in personalized cancer models

  • Co-culture systems to examine PXMP4's role in tumor-stroma interactions