Recombinant Bovine Peroxisomal biogenesis factor 3 (PEX3)

Basic Research Questions

• What is the role of PEX3 in peroxisome biogenesis?

PEX3 functions as a master regulator of peroxisome biogenesis across species. It is an integral peroxisomal membrane protein that serves as a docking receptor for PEX19, which is the cytosolic chaperone for peroxisomal membrane proteins (PMPs). This interaction initiates the biogenesis of peroxisome precursors from the endoplasmic reticulum (ER) . PEX3 assembles membrane vesicles before matrix proteins are translocated, making it essential for the early stages of peroxisome formation . Research has demonstrated that cells lacking PEX3 are unable to form normal peroxisomes, but reintroduction of functional PEX3 restores peroxisome biogenesis .

Methodological approach: To study PEX3's role in peroxisome biogenesis, researchers can employ PEX3 knockout/knockdown systems followed by complementation with recombinant PEX3. Fluorescence microscopy with peroxisomal markers allows visualization of peroxisome reconstitution. For example, in Hansenula polymorpha pex3 cells, reintroduction of wild-type Pex3p led to the rapid reappearance of one small peroxisome per cell, demonstrating its essential role in de novo peroxisome formation .

• How does PEX3 interact with PEX19, and what are the critical residues involved?

The PEX3-PEX19 interaction is conserved across species and represents a crucial step in peroxisome biogenesis. Specific amino acid residues in the PEX19-binding domain of PEX3 are required for this interaction. Studies using site-directed mutagenesis have identified several conserved residues that are essential for PEX19 binding.

Methodological data: Yeast two-hybrid assays and pull-down experiments have shown that:

• In trypanosomal PEX3 (TbPex3), mutation F102A completely abolished interaction with TbPex19

• Similarly, L105A mutation in TbPex3 eliminated PEX19 binding

• The corresponding leucine residue in human PEX3 (L107) has been confirmed to contribute to PEX19 binding

• Mutations outside the immediate binding region (such as TbPex3-K98A) did not affect interaction

• What is the subcellular distribution and localization pattern of PEX3?

PEX3 primarily localizes to peroxisomal membranes but has also been observed in the ER during early stages of peroxisome biogenesis.

Methodological approach: Immunofluorescence microscopy with specific antibodies against PEX3 and other organelle markers reveals that:

• PEX3 colocalizes with peroxisomal markers such as PEX14 and ABCD3

• PEX3 shows variable abundance across different cell types within the same tissue

• In kidney tissue, PEX3 is highly abundant in proximal tubules compared to distal tubules

• During peroxisome biogenesis, PEX3 may temporarily localize to reticular structures devoid of peroxisomal matrix proteins

Subcellular fractionation studies confirm that PEX3 co-fractionates with peroxisomal enzymes and, to a lesser extent, with ER markers such as BiP, supporting its dual localization during peroxisome formation .

Advanced Research Questions

• What experimental systems and methodologies are optimal for studying recombinant PEX3 function?

Several experimental systems have been successfully employed to study PEX3 function, each with specific advantages for different research questions.

Methodological approach:

For recombinant protein studies specifically:

• E. coli expression systems are suitable for producing domains of PEX3 for structural studies

• Insect cell or mammalian expression systems may better preserve folding and post-translational modifications

• When using PEX3-GFP fusion proteins, verify that peroxisomal protein import is not impaired

• How does overexpression of PEX3 affect peroxisome dynamics and what mechanisms underlie these effects?

Interestingly, while PEX3 is essential for peroxisome biogenesis, high levels of PEX3 expression can induce peroxisome degradation through a selective autophagy process called pexophagy.

Methodological findings:

• In PEX3-loaded cells, peroxisomes become ubiquitinated, clustered, and subsequently degraded in lysosomes

• This degradation process requires peroxisomal targeting of PEX3

• The autophagic receptor protein NBR1 is essential for this PEX3-induced pexophagy

• Another autophagic receptor, SQSTM1/p62, is required for clustering but not degradation of peroxisomes

• The degradation of peroxisomes can be inhibited by treatment with autophagy inhibitors such as 3-methyladenine and bafilomycinA

This dual role of PEX3 in both biogenesis and degradation suggests a regulatory function in peroxisome homeostasis, which researchers should consider when designing experiments with recombinant PEX3.

• What approaches can be used to study the structure-function relationship of PEX3 and identify critical functional domains?

Understanding the structure-function relationship of PEX3 is crucial for elucidating its mechanism of action and developing potential therapeutic strategies.

Methodological approaches:

• Site-directed mutagenesis: Target conserved residues identified through sequence alignment. For example:

  • Mutation of the PEX19-binding region (such as F102A in TbPex3 or the corresponding W128A in S. cerevisiae Pex3) abolishes interaction with PEX19

• Domain truncation/deletion analysis:

  • Expression of the first 50 amino acids of Pex3p in H. polymorpha resulted in vesicle formation from the nuclear envelope

  • N-terminal and C-terminal truncations can help map functional regions

• Epitope tagging and fusion proteins:

  • Myc-tagging at different positions (N-terminus, C-terminus, or internal regions) can help determine which regions tolerate modifications

  • GFP fusion proteins can be used to track localization and dynamics

• Chimeric protein analysis:

  • Swapping domains between PEX3 from different species can identify conserved functional modules

• Cross-linking and proximity labeling:

  • To identify transient interaction partners during different stages of peroxisome biogenesis

• How can species-specific differences in PEX3 be leveraged for therapeutic or experimental applications?

The significant sequence divergence between PEX3 proteins from different species presents opportunities for species-specific targeting, particularly relevant for parasitic diseases and selective inhibitor development.

Methodological findings:

• Trypanosomal PEX3 (TbPex3) shares only 7% amino acid identity with human PEX3, despite maintaining functional conservation of the PEX19-binding domain

• This limited sequence similarity makes TbPex3 an attractive therapeutic target for diseases caused by trypanosomatids

• A small molecule screen identified compounds that preferentially inhibit the interaction between TbPex3 and TbPex19 versus human counterparts

• Administration of such compounds to T. brucei led to compromised glycosome biogenesis and was lethal to both procyclic and bloodstream forms of the parasite at concentrations that had limited effect on human cells

This approach demonstrates how understanding species-specific differences in PEX3 can be leveraged for drug development against parasitic diseases while minimizing off-target effects on host peroxisomes.

• What are the challenges in expressing and purifying functional recombinant PEX3, and how can they be addressed?

Expressing and purifying functional PEX3 presents several challenges due to its membrane protein nature.

Methodological solutions:

For crystallization studies, researchers have successfully used truncated versions of PEX3 lacking the N-terminal transmembrane segment while maintaining the PEX19-binding capacity .

• How does PEX3 function in different tissues, and what are the tissue-specific requirements for PEX3?

PEX3 expression and function vary across different tissues and cell types, with tissue-specific requirements revealed by recent studies.

Methodological findings:

• Immunohistochemical studies in mouse tissues show variable PEX3 abundance:

  • Highly abundant in kidney proximal tubules

  • Less abundant in distal tubules

  • Variable expression in other tissues

• Tissue-specific knockout studies reveal differential requirements:

  • Germ cell-specific deletion of Pex3 in mice results in male sterility

  • The same deletion leads to destruction of intercellular bridges between spermatids and formation of multinucleated giant cells

  • Sertoli cell-specific deletion of Pex3 does not affect spermatogenesis

  • Proteomic analysis of Pex3-deleted spermatids reveals defective expressions of peroxisomal proteins and spermiogenesis-related proteins

These findings suggest that PEX3-dependent peroxisome function has tissue-specific roles that may not be universally critical across all cell types.

• How is PEX3 regulated by cell signaling pathways, and how can this knowledge be applied in experimental designs?

Recent research has revealed interconnections between PEX3 function and cellular signaling pathways, particularly protein kinases that regulate peroxisome biogenesis.

Methodological findings:

• Protein Kinase C (PKC) positively regulates peroxisome biogenesis by promoting peroxisome-ER interaction

• PKC inhibits GSK3β, which normally negatively regulates peroxisome-ER tethering

• This promotes ACBD5-VAPB interaction, which is critical for peroxisome biogenesis

• Small molecule kinase inhibitor screening revealed multiple regulators of peroxisome abundance:

  • PKC inhibitors reduced peroxisome numbers

  • GSK3β inhibitors increased peroxisome numbers

Implications for experimental design:

• Consider the impact of cell signaling status when studying PEX3 function

• Control for or exploit signaling pathways when manipulating peroxisome biogenesis

• Potential for pharmacological modulation of peroxisome numbers through targeting these pathways

These insights provide new avenues for controlling peroxisome biogenesis in experimental systems through manipulation of signaling pathways that regulate PEX3 function.

Disease-Related Research Questions

• What are the implications of PEX3 mutations in human disease, and how can recombinant PEX3 be used in disease models?

Mutations in PEX3 are associated with peroxisome biogenesis disorders (PBDs), particularly Zellweger syndrome of complementation group G (CG-G).

Methodological approaches:

• Patient fibroblasts with PEX3 mutations can be complemented with recombinant wild-type PEX3 to restore peroxisome biogenesis

• A documented case (patient PBDG-02) carried a homozygous 97-bp deletion resulting in a 32-amino-acid truncation and a frameshift

• Genomic analysis revealed a T→G mutation at the splicing site boundary of intron 10 and exon 11, leading to deletion of exon 11

• Expression of wild-type human PEX3 cDNA morphologically and biochemically restored peroxisome biogenesis in these patient fibroblasts

Disease models can be developed using:

• CRISPR/Cas9 to generate PEX3 knockout cell lines mimicking complete loss of function

• Introduction of patient-specific mutations to study partial loss of function

• Conditional knockout animal models to study tissue-specific effects

Such models are valuable for testing therapeutic approaches, including gene therapy, small molecule screening, and chaperone-mediated stabilization of mutant PEX3.

• How can evolutionary conservation and divergence in PEX3 inform both basic research and therapeutic development?

The evolutionary pattern of PEX3 across species reveals both conserved functional domains and significant sequence divergence, providing insights for research and therapeutic strategies.

Methodological considerations:

The identification of trypanosomal PEX3 (TbPex3) illustrates this approach:

• It was discovered through HHpred bioinformatics platform, which analyzes protein secondary structure rather than primary sequence

• Despite only 7% amino acid identity with human PEX3, it maintains the Pex19 interaction domain

• This divergence enabled development of small molecule inhibitors that selectively disrupt the TbPex3-TbPex19 interaction

• Such inhibitors were lethal to T. brucei while having limited effects on human cells