Recombinant Macaca fascicularis Peroxisomal biogenesis factor 3 (PEX3)

What is the structure and function of Macaca fascicularis PEX3?

Macaca fascicularis PEX3 is a 373-amino acid peroxisomal transmembrane protein with its C-terminus facing the cytosol. The protein sequence begins with "mLRSVWNFLKRHKKKCIFLGTVLGGVYILGKYGQKKIREIQEREAAEYIAQARRQYHFES" and contains distinctive structural domains responsible for various cellular interactions . PEX3 functions as a conserved multifunctional peroxisomal protein involved in several critical processes including peroxisomal membrane protein insertion, pexophagy (selective peroxisome degradation), and the formation of membrane contact sites between peroxisomes and other organelles . The protein is typically stored in a Tris-based buffer with 50% glycerol to maintain stability, and it's best stored at -20°C or -80°C for extended periods to preserve its functional properties .

What experimental systems are available for studying Macaca fascicularis PEX3?

Research on Macaca fascicularis PEX3 benefits from several established experimental systems. Commercially available recombinant proteins with various tag options facilitate in vitro studies . For cell-based research, the sequenced Macaca fascicularis genome enables precise genetic manipulation and expression analysis, with species-specific gene expression microarrays available for transcriptomic studies . Additionally, primary cell cultures from Macaca fascicularis tissues provide physiologically relevant experimental systems. For subcellular localization studies, fluorescently tagged PEX3 can be used for live-cell imaging, though careful consideration must be given to tag placement to avoid disrupting protein function, as the C-terminus faces the cytosol . For protein interaction studies, proximity biotinylation approaches using TurboID-tagged PEX3 have proven effective in identifying proteins in the molecular microenvironment of PEX3 .

How does PEX3 regulate the formation of inter-organelle contact sites?

PEX3 plays a crucial role in establishing and maintaining contact sites between peroxisomes and other organelles. Research has shown that elevated PEX3 levels in Saccharomyces cerevisiae induce the formation of peroxisome clusters surrounded by lipid droplets, facilitated by both peroxisome-peroxisome and peroxisome-lipid droplet contact sites . The cytosolic domain of PEX3 appears to directly bind peroxisomes, suggesting a direct role in homotypic contact site formation. Interestingly, these clustering phenomena occur independently of known PEX3 partners in other processes, including Pex19, Inp1, and Atg36 .

For peroxisome-lipid droplet contact sites, the lipid droplet-localized triacylglycerol lipase Tgl4 is required and becomes enriched at this interface along with other lipases . This suggests a functional role for these contact sites in facilitating fatty acid transfer between compartments. In Hansenula polymorpha, PEX3 is enriched at peroxisome-vacuole contact sites, which expand when cells transition from glucose to methanol metabolism—a shift requiring peroxisomal expansion . The different contact site preferences in different yeast species suggest that PEX3's multiple functions may have evolved sequentially, resulting in species-specific adaptations in contact site formation.

What is the molecular mechanism of PEX3's role in pexophagy regulation?

PEX3 functions beyond simply serving as a docking site for pexophagy receptors—it actively regulates the initiation of selective peroxisome degradation. In Pichia pastoris, PEX3 interacts with the pexophagy receptor Atg30 and contributes significantly to pexophagy signaling . Specific domains on PEX3, particularly regions containing the amino acid residues L320 and N325, are responsible for this interaction . When these residues are mutated (as in pex3 L320P and pex3 N325D), the interaction with Atg30 is disrupted, and pexophagy is impaired .

PEX3 plays a dual role in this process: it not only recruits Atg30 to peroxisomes under proliferation conditions but also activates Atg30 via phosphorylation—a prerequisite for Atg30's interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8 . Additionally, PEX3 facilitates the recruitment of Atg11 to the receptor protein complex. This reveals a sophisticated regulatory mechanism where PEX3 serves as both a scaffold and an activator in the pexophagy signaling pathway.

How does overexpression of PEX3 affect peroxisome morphology and function?

Overexpression of PEX3 dramatically alters peroxisome morphology, distribution, and interactions with other organelles. In Saccharomyces cerevisiae, PEX3 overexpression from a strong constitutive promoter causes a striking reduction in peroxisome number, from an average of 5.5 structures per cell to predominantly one structure . This single structure is frequently (in 55% of cells) found in close proximity to the vacuole .

Similarly, in Drosophila melanogaster, PEX3 overexpression in the midgut reduces peroxisome number while increasing their size, a phenotype that becomes more pronounced during development . In adult Drosophila midgut, the effect is particularly dramatic, with a substantial increase in both peroxisomal and lipid droplet size .

High-resolution imaging reveals that these enlarged peroxisomes are closely associated with lipid droplets, evidenced by the deformation of organelle shapes at their contact sites . The observation of similar phenotypes across evolutionary distant species (yeast and fruit flies) suggests that this role of PEX3 in regulating peroxisome morphology and organelle contacts is a conserved function . These changes likely impact metabolic functions, particularly lipid metabolism, given the close association with lipid droplets.

What are the optimal approaches for studying PEX3-mediated protein interactions?

To effectively study PEX3-mediated protein interactions, researchers should employ a complementary set of techniques. Proximity-based approaches such as BioID or TurboID labeling (as demonstrated with Pex3-TurboID in yeast ) are particularly valuable for identifying proteins in the native cellular environment of PEX3. When using these methods, researchers should consider the orientation of the tag, as the C-terminus of PEX3 faces the cytosol .

For direct interaction studies, co-immunoprecipitation followed by western blotting or mass spectrometry can identify stable binding partners. In vitro binding assays using purified recombinant Macaca fascicularis PEX3 can determine whether interactions are direct and can quantify binding affinities. Surface plasmon resonance or isothermal titration calorimetry would be appropriate for these quantitative measurements.

For visualizing interactions in live cells, researchers should consider fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC). When designing these experiments, it's crucial to include appropriate controls, such as PEX3 mutants with specific interaction domains disrupted (e.g., the L320P and N325D mutations that affect Atg30 binding ).

How should researchers design experiments to investigate the role of PEX3 in membrane contact site formation?

Investigating PEX3's role in membrane contact site formation requires a multifaceted experimental approach. First, researchers should establish reliable markers for visualizing peroxisomes and potential partner organelles. Fluorescently-tagged markers such as mCherry-SKL (for peroxisome lumen) and markers for Pex3 itself or Pex14 have proven effective . For studying the recruitment of PEX3 to contact sites, super-resolution microscopy techniques like Airyscan confocal microscopy are recommended to achieve the resolution necessary to visualize these structures .

For manipulating PEX3 levels, researchers should consider inducible expression systems to control the timing and level of expression. The choice of promoter is critical—studies have used strong constitutive promoters like TEF1 to achieve overexpression . When interpreting results, it's important to compare observations across multiple experimental systems to distinguish conserved from species-specific effects, as demonstrated by the comparative studies in yeast and Drosophila .

To identify molecular requirements for contact site formation, systematic mutagenesis of PEX3 domains should be performed, followed by functional assays to assess contact site formation. Additionally, deletion of potential partner proteins (such as Tgl4 for peroxisome-lipid droplet contacts ) can help determine the molecular requirements for different types of contact sites.

What controls and validation steps are necessary when studying the effects of PEX3 mutations?

When investigating the effects of PEX3 mutations, several critical controls and validation steps must be implemented. First, expression levels of mutant proteins should be confirmed to be comparable to wild-type levels to avoid misinterpreting overexpression or underexpression artifacts. Western blotting against PEX3 or against relevant tags is appropriate for this purpose.

Second, proper localization of mutant PEX3 proteins to peroxisomes should be verified through fluorescence microscopy or subcellular fractionation. This is essential because mislocalization could cause indirect effects unrelated to the specific function being studied.

Third, when analyzing specific functions like pexophagy, multiple independent assays should be employed. For example, the study that identified the L320P and N325D mutations affecting pexophagy used a GFP-PTS1 degradation assay to monitor peroxisome turnover . Additional functional assays might include measuring biochemical activities associated with peroxisomes or assessing peroxisome numbers and morphology.

Finally, complementation studies should be performed to confirm that the observed phenotypes are directly attributable to the PEX3 mutations. This approach was effectively used to characterize the pexophagy defects of specific pex3 mutants, demonstrating that the L320P and N325D mutations specifically affected pexophagy without disrupting other peroxisomal functions .

How can researchers differentiate between direct and indirect effects when manipulating PEX3?

Distinguishing direct from indirect effects of PEX3 manipulation requires a systematic experimental approach. First, researchers should implement time-course experiments following PEX3 manipulation to establish the temporal sequence of events—effects appearing immediately are more likely to be direct consequences of PEX3 function. Inducible expression systems are valuable for this purpose.

Second, structure-function analysis using specific PEX3 mutants can help delineate which phenotypes are linked to specific domains and functions. This approach was successfully employed with the L320P and N325D mutations, which specifically disrupted pexophagy without affecting other peroxisomal functions . When combined, these mutations (pex3 L320P,N325D) produced an even stronger pexophagy phenotype, further supporting a direct role for PEX3 in this process .

Third, in vitro reconstitution of PEX3-dependent activities with purified components can provide strong evidence for direct effects. For example, demonstrating that purified recombinant Macaca fascicularis PEX3 can directly bind and activate potential partner proteins in the absence of other cellular components would support a direct functional relationship.

How should contradictory data about PEX3 function from different model systems be reconciled?

When faced with contradictory data about PEX3 function from different model systems, researchers should consider several factors. First, evolutionary divergence may have resulted in species-specific adaptations of PEX3 function. This is exemplified by the different contact sites formed upon PEX3 overexpression in different yeast species—peroxisome-vacuole contacts in Hansenula polymorpha versus peroxisome-peroxisome and peroxisome-lipid droplet contacts in Saccharomyces cerevisiae .

Second, methodological differences should be systematically examined. Variations in expression levels, protein tagging strategies, growth conditions, and analytical methods can all contribute to apparently contradictory results. Standardizing these parameters across studies can help identify true biological differences versus technical artifacts.

Third, direct comparative studies using identical experimental approaches across different model systems provide the most reliable basis for comparison. The study examining PEX3 overexpression in both yeast and Drosophila exemplifies this approach, revealing conserved aspects of PEX3 function in peroxisome morphology regulation and organelle contact site formation .

Finally, researchers should develop integrative models that accommodate species-specific variations while identifying conserved core functions. Such models should explicitly address how evolutionary, metabolic, or physiological differences between model systems might explain the observed functional variations in PEX3.

What statistical approaches are recommended for analyzing phenotypic changes in PEX3 studies?

Analyzing phenotypic changes in PEX3 studies requires robust statistical approaches tailored to the specific data types. For quantitative measurements such as peroxisome number, size, or distribution, parametric tests (t-tests or ANOVA) are appropriate if the data follow a normal distribution. If normality cannot be assumed, non-parametric alternatives should be used.

For microscopy-based analyses of peroxisome morphology or contact sites, quantitative image analysis tools should be employed to extract objective measurements from sufficient numbers of cells. The study examining PEX3 overexpression effects quantified peroxisome numbers per cell in control versus overexpression conditions, providing a statistical basis for the observation that overexpression reduces peroxisome number .

When analyzing complex phenotypes involving multiple variables, multivariate statistical approaches may be necessary. For example, principal component analysis could help identify patterns in data sets that include measurements of peroxisome number, size, proximity to other organelles, and biochemical activities.

Importantly, sample sizes should be sufficiently large to ensure statistical power, and appropriate controls should be included in all analyses. Multiple independent biological replicates are essential to ensure reproducibility. When reporting results, both the effect size and statistical significance should be presented to provide a complete picture of the biological relevance of the findings.

How does Macaca fascicularis PEX3 compare to human PEX3, and what are the implications for translational research?

Macaca fascicularis PEX3 shares high sequence similarity with human PEX3, reflecting the close evolutionary relationship between these primates. The Macaca fascicularis genome project has identified approximately 17,387 orthologs of human protein-coding genes , and PEX3 is likely among these conserved genes. This high degree of conservation makes Macaca fascicularis an excellent model for studying peroxisome biology relevant to human health and disease.

The detailed sequence and functional characterization of Macaca fascicularis PEX3 provides a foundation for comparative studies with human PEX3 . Such studies could reveal subtle species differences that might affect drug interactions or disease mechanisms. For translational research, understanding these similarities and differences is crucial when using Macaca fascicularis as a model for human peroxisomal disorders.

The availability of recombinant Macaca fascicularis PEX3 for research applications facilitates detailed biochemical and structural studies that can inform our understanding of human PEX3 function. Additionally, the genome-based analysis of Macaca fascicularis enables high-resolution genotyping and microarray-based gene expression profiling for animal stratification in research studies , enhancing the translational value of this model system.

What are the advantages of using Macaca fascicularis models for studying peroxisomal disorders compared to other model organisms?

Macaca fascicularis offers several distinct advantages for studying peroxisomal disorders. As a nonhuman primate, it shares higher genetic similarity with humans than rodent or yeast models, making it particularly valuable for translational research. The Macaca fascicularis genome has been fully sequenced, enabling precise genetic manipulation and analysis .

Unlike rhesus macaques that have seasonal fertility, Macaca fascicularis lacks this limitation, making it more suitable for reproductive toxicity studies that might be relevant for peroxisomal disorders affecting development . This is particularly important since many peroxisomal biogenesis disorders have developmental components.

For drug development and safety testing related to peroxisomal disorders, Macaca fascicularis is the most widely used primate species . Validated assays for measuring blood parameters and safety biomarkers such as liver enzymes are readily available , facilitating comprehensive physiological assessments that may be relevant to peroxisomal function.

Furthermore, the availability of species-specific gene expression microarrays designed based on the predicted transcripts from the Macaca fascicularis genome enables detailed transcriptomic studies of peroxisome-related genes. This provides a powerful tool for investigating molecular mechanisms and potential therapeutic targets in peroxisomal disorders.

How can studies of Macaca fascicularis PEX3 inform our understanding of peroxisome-related diseases?

Studies of Macaca fascicularis PEX3 can significantly advance our understanding of peroxisome-related diseases through several mechanisms. First, detailed characterization of PEX3 structure and function in this primate model provides insights into fundamental peroxisomal processes that may be disrupted in human disease . The multi-functional nature of PEX3—involved in peroxisomal membrane protein insertion, pexophagy, and contact site formation—means that alterations in this protein could have diverse pathological consequences.

Second, the role of PEX3 in regulating contact sites between peroxisomes and other organelles, particularly lipid droplets , has implications for disorders of lipid metabolism. The observation that PEX3 overexpression affects the distribution and morphology of both peroxisomes and lipid droplets suggests that proper PEX3 function is critical for lipid homeostasis . Dysregulation of these processes could contribute to metabolic disorders.

Third, the involvement of PEX3 in pexophagy signaling highlights its potential role in quality control mechanisms for peroxisomes. Defects in these mechanisms could lead to accumulation of damaged peroxisomes or insufficient peroxisome numbers, both of which could have pathological consequences.

Finally, the high genetic similarity between Macaca fascicularis and humans means that insights gained from studying PEX3 in this model are likely to be directly relevant to human peroxisomal disorders. This makes Macaca fascicularis PEX3 an invaluable research tool for understanding the molecular mechanisms of peroxisomal diseases and developing potential therapeutic strategies.