Recombinant Pongo abelii Peroxisomal membrane protein 11B (PEX11B)

What is the structural organization of PEX11B in the peroxisomal membrane?

PEX11B is an integral membrane protein with a specific topology that facilitates its function in peroxisomal proliferation. Studies using epitope-specific antibodies and protease protection assays have revealed that PEX11B contains two transmembrane domains that flank an internal region exposed to the peroxisomal matrix. Both the N-terminus and C-terminus of the protein face the cytosol, which is critical for its interactions with cytosolic factors . The protein contains 259 amino acids in Pongo abelii (Sumatran orangutan), with a complete amino acid sequence that begins with MDAWVRFSAQ and continues through specific structural regions that contribute to its function . This topological arrangement positions PEX11B optimally to mediate membrane remodeling during peroxisome proliferation.

How does the molecular structure of PEX11B contribute to its functional capabilities?

PEX11B contains several structural elements critical for its role in peroxisome membrane dynamics. Most notably, an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids has been demonstrated as crucial for membrane elongation and self-interaction of the protein . This amphipathic helix likely inserts into the peroxisomal membrane, inducing curvature that facilitates membrane elongation. Additionally, PEX11B contains a glycine-rich internal region that research has shown to be dispensable for peroxisome membrane elongation and division processes . The protein's structure includes regions that mediate interaction with PEX19, a peroxisomal membrane protein import receptor, which is essential for proper localization and function . Understanding these structural features provides critical insight into how PEX11B facilitates peroxisome proliferation.

What experimental methods are recommended for optimal storage and handling of recombinant PEX11B?

For researchers working with recombinant Pongo abelii PEX11B, proper storage and handling are critical for maintaining protein integrity and experimental reproducibility. The protein should be stored in a Tris-based buffer with 50% glycerol, which has been optimized for this specific protein . For long-term storage, the protein should be kept at -20°C, while extended storage periods necessitate conservation at -20°C or -80°C . Working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw cycles . It is strongly recommended to avoid repeated freezing and thawing as this can lead to protein denaturation and loss of activity . For experimental protocols requiring multiple uses, researchers should prepare small aliquots of the protein upon receipt to preserve functionality across experiments.

How does PEX11B self-interaction regulate peroxisomal membrane dynamics?

PEX11B self-interaction represents a critical regulatory mechanism for controlling peroxisomal membrane deformation. Research has demonstrated that this self-interaction strongly depends on the detergent conditions used during protein solubilization, suggesting that the interaction is sensitive to the lipid environment . The N-terminal amphipathic helix (Helix 2) within the first 40 amino acids plays an essential role in facilitating this self-interaction, as mutations in this region disrupt both self-interaction and membrane elongation capabilities . The current model suggests that PEX11B monomers assemble into oligomeric structures that generate or stabilize membrane curvature during peroxisome elongation. This self-interaction appears to work in conjunction with membrane lipids to regulate the protein's membrane deforming activity . Experimental approaches to study this phenomenon typically employ co-immunoprecipitation under varying detergent conditions, FRET analysis, or crosslinking studies to capture these interactions in their native state.

What role do the N-terminal domains of PEX11B play in peroxisomal membrane remodeling?

The N-terminal region of PEX11B contains critical functional elements that drive peroxisomal membrane remodeling. Research has established that an amphipathic helix (Helix 2) within the first 40 amino acids is essential for membrane elongation functions . This helix likely inserts into the peroxisomal membrane at an angle that induces membrane curvature, thereby initiating the elongation process necessary for subsequent division. Interestingly, research has shown that N-terminal cysteines are not essential for membrane elongation, despite their conservation across species . Additionally, putative N-terminal phosphorylation sites have been demonstrated to be dispensable for PEX11B function . These findings highlight the specialized nature of the N-terminal amphipathic helix in membrane remodeling, while suggesting that other conserved elements may serve secondary or species-specific functions. Researchers investigating these domains typically employ site-directed mutagenesis followed by functional assays measuring peroxisome elongation and division efficiency.

How do mutations in PEX11B contribute to peroxisome biogenesis disorders?

Mutations in PEX11B have been associated with a distinct form of peroxisome biogenesis disorder (PBD-14B), which presents with a unique clinical profile. Research has identified several loss-of-function mutations including homozygous nonsense mutations (c.235C>T p.(Arg79Ter), c.136C>T p.(Arg46Ter)), compound heterozygous mutations (c.595C>T p.(Arg199Ter)), and deletions (PEX11B ex1-3 del) . These mutations disrupt PEX11B's ability to facilitate peroxisome membrane elongation and division, leading to abnormal peroxisome morphology and dynamics . The clinical manifestations include bilateral congenital cataracts as a consistent early feature, along with variable presentations of short stature, skeletal abnormalities, and dysmorphism . Biochemically, patients may exhibit very low plasmalogen levels and mildly deranged very long chain fatty acid profiles, reflecting impaired peroxisomal function . Unlike classic peroxisome biogenesis disorders such as Zellweger syndrome, PEX11B-related disorders tend to present with more moderate symptoms, suggesting that some peroxisomal functions remain partially intact.

What are the optimal methods for detecting PEX11B mutations in clinical samples?

Detection of PEX11B mutations requires a comprehensive approach that combines multiple molecular techniques. Next-generation sequencing (NGS) has proven effective for identifying point mutations, small insertions, and deletions in PEX11B . For larger genomic rearrangements that may be missed by standard sequencing, techniques such as ExomeDepth analysis of NGS data followed by confirmation with quantitative PCR (qPCR) or droplet digital PCR (ddPCR) are recommended . In one research approach, primers for PEX11B screening were designed to amplify and sequence all coding exons plus intron-exon boundaries according to RefSeq transcript NM_003846 . For family studies, autozygosity mapping can be particularly valuable when analyzing affected sibling pairs . A comprehensive diagnostic approach should include both sequence analysis for point mutations and dosage analysis for copy number variants. This multi-faceted strategy ensures detection of the full spectrum of potential PEX11B mutations, from single nucleotide variants to larger genomic rearrangements that might otherwise be missed.

What biochemical assays are most informative for characterizing peroxisomal dysfunction in PEX11B-related disorders?

Biochemical profiling provides critical insights into the functional consequences of PEX11B mutations and helps characterize the severity of peroxisomal dysfunction. The most informative assays include plasmalogen quantification, as very low plasmalogen levels have been observed in some PEX11B-deficient patients . Additionally, very long chain fatty acid (VLCFA) profiling can reveal mild abnormalities in fatty acid metabolism . These biochemical markers reflect disruptions in peroxisomal lipid metabolism pathways that are secondary to defects in peroxisome division and proliferation. A comprehensive biochemical evaluation should include analysis of plasmalogens (particularly C16:0 and C18:0 species), VLCFAs (C22:0, C24:0, C26:0, and their ratios to C22:0), phytanic and pristanic acids, and bile acid intermediates. The pattern of biochemical abnormalities can help distinguish PEX11B-related disorders from other peroxisomal biogenesis disorders and guide treatment approaches. Importantly, these biochemical signatures may vary between patients with different PEX11B mutations, reflecting genotype-phenotype correlations.

How can researchers effectively study PEX11B membrane topology in experimental settings?

Investigating PEX11B membrane topology requires combining complementary approaches to generate a comprehensive understanding of protein orientation. Epitope-specific antibody studies represent a powerful technique, wherein antibodies targeted to different regions of PEX11B are used to determine which portions are accessible from which side of the membrane . These studies can be complemented with protease protection assays, where intact peroxisomes are treated with proteases to cleave exposed protein regions while membrane-embedded or matrix-facing regions remain protected . For more detailed structural analysis, researchers can employ selective permeabilization of either the peroxisomal or plasma membrane followed by immunofluorescence microscopy to determine epitope accessibility. Additionally, cysteine scanning mutagenesis coupled with membrane-impermeable sulfhydryl reagents can identify transmembrane domains with high precision. These techniques have collectively established that PEX11B contains two transmembrane domains flanking an internal region exposed to the peroxisomal matrix, with both N- and C-termini facing the cytosol .

How is PEX11B conserved across different species, and what does this reveal about its evolutionary importance?

PEX11B demonstrates remarkable evolutionary conservation across diverse species, highlighting its fundamental importance in peroxisome biology. The gene has been identified in mammals including humans, mice, rats, sheep, domestic guinea pigs, naked mole-rats, dogs, cats, and cows, as well as in non-mammalian vertebrates such as zebrafish and even plants like Thale Cress . This broad conservation suggests that PEX11B emerged early in eukaryotic evolution and has maintained its essential function in peroxisome dynamics. Sequence alignment of PEX11B across species reveals conservation of key functional domains, particularly the N-terminal amphipathic helix that is crucial for membrane elongation and self-interaction . The amino acid sequence of Pongo abelii (Sumatran orangutan) PEX11B shows high homology with human PEX11B, making it a valuable model for studying human peroxisome disorders . This evolutionary conservation provides researchers with the opportunity to use various model organisms to study PEX11B function, with findings likely translatable across species. The maintained presence of PEX11B throughout eukaryotic evolution underscores its indispensable role in peroxisome biogenesis and cellular metabolism.

What are the species-specific differences in PEX11B function that researchers should consider when designing experiments?

While PEX11B is highly conserved across species, researchers must account for subtle but potentially significant species-specific differences when designing experiments. Comparative genomic analyses have identified variations in regulatory regions that may affect expression patterns, potentially leading to differences in peroxisome abundance and morphology between species . Additionally, interaction partners of PEX11B may vary across species, potentially altering the regulatory networks controlling peroxisome proliferation. When using model organisms, researchers should validate key findings in human cells to ensure translational relevance. In particular, the biochemical consequences of PEX11B dysfunction may manifest differently across species due to variations in peroxisomal metabolic pathways. For instance, plants utilize peroxisomes for additional specialized metabolic functions not present in animals . Experimental design should include appropriate species-specific controls and consider using multiple model systems to validate findings. When working with recombinant PEX11B from different species (such as Pongo abelii), researchers should evaluate protein interactions with species-matched binding partners whenever possible to avoid artifacts arising from inter-species compatibility issues.