Recombinant Human Peroxisomal membrane protein 11B (PEX11B)

What is PEX11B and what are its primary functions in human cells?

PEX11B (Peroxisomal Biogenesis Factor 11 Beta) is a protein-coding gene that produces a membrane protein found in the peroxisomal membrane. It serves several critical functions in peroxisomal dynamics, primarily facilitating peroxisomal proliferation through interaction with PEX19 . The protein encoded by PEX11B plays a crucial role in multiple aspects of peroxisomal growth and division, including:

• Membrane deformation and elongation of peroxisomes

• Recruitment of mitochondrial fission factor (MFF) and FIS1 to constriction sites

• Activation of the dynamin-related protein 1 (DRP1) required for membrane fission

How does PEX11B differ from other PEX11 isoforms?

Humans express three PEX11 isoforms: PEX11α, PEX11β, and PEX11γ. While these isoforms share some structural similarities, they exhibit notable functional differences:

• PEX11β appears to have a more fundamental role in peroxisome division, as neither PEX11α nor PEX11γ can fully complement the loss of PEX11β function

• The promoter regions of PEX11α and PEX11β contain different binding sites, suggesting they are regulated by distinct pathways

• PEX11β has a functional SMAD2/3 binding site in its promoter, linking it to TGFβ signaling pathways, which is not observed in other PEX11 isoforms

• PEX11β exhibits a striped distribution pattern on peroxisomal membranes that does not coincide with other peroxisomal proteins like Pex14p and PTS1 matrix proteins

The three isoforms likely evolved to respond to different cellular cues and environmental conditions, allowing for specialized regulation of peroxisome dynamics based on cell type and metabolic state.

What are the major cellular pathways associated with PEX11B?

PEX11B functions within several important cellular pathways:

• Peroxisomal lipid metabolism pathway - PEX11B plays a crucial role in the proliferation of peroxisomes, which are essential organelles for lipid metabolism

• TGFβ signaling pathway - The presence of a functional SMAD2/3 binding site in the PEX11β promoter establishes a novel link between TGFβ signaling and peroxisome proliferation

• PPARγ signaling - Research indicates that PPARγ may regulate PEX11B expression, as evidenced by studies showing PPARγ-dependent effects of PEX11B on neural differentiation

• SIRT1 interaction pathway - PEX11B appears to interact with SIRT1 during human embryonic stem cell (hESC) neural differentiation, with SIRT1 inhibition counteracting the effects of PEX11B knockdown

These interconnected pathways highlight PEX11B's integrated role in cellular metabolism, stress response, and developmental processes.

What are the molecular mechanisms by which PEX11B induces peroxisomal membrane deformation?

PEX11B induces peroxisomal membrane deformation through several coordinated molecular mechanisms:

• Amphipathic helix-mediated membrane bending: The N-terminal amphipathic helix of PEX11β appears to be crucial for membrane deformation. Point mutations in this region significantly reduce peroxisomal fission and decrease oligomer formation, suggesting this structural element directly interacts with the lipid bilayer to induce curvature .

• Oligomerization at constriction sites: Reconstituted PEX11β protein localizes to membrane constriction sites in proteo-liposomes. This localization pattern suggests that PEX11β oligomers form at specific regions of the peroxisomal membrane to initiate the deformation process .

• Recruitment of fission machinery: After initial membrane deformation, PEX11β recruits the necessary division machinery, including DLP1 (dynamin-like protein 1) and Mff (mitochondrial fission factor). Knockdown studies have shown that both DLP1 and Mff are required for PEX11β-mediated peroxisomal fission, as their absence results in elongated, undivided peroxisomes even when PEX11β is overexpressed .

• Sequential segmentation pattern: Peroxisomal markers exhibit a "beads on a string" appearance in fission-deficient peroxisomes, indicating that PEX11β may establish segmentation patterns before complete fission occurs .

The combination of direct membrane interaction, protein clustering, and recruitment of division factors enables PEX11B to effectively remodel peroxisomal membranes and drive organelle division.

How do mutations in PEX11B contribute to the pathogenesis of peroxisome biogenesis disorders?

Mutations in PEX11B have been linked to peroxisome biogenesis disorder 14B (PBD14B), a condition with varying clinical manifestations. The pathogenic mechanisms include:

• Loss of peroxisome division capability: Loss-of-function mutations in PEX11B (including nonsense mutations like c.235C>T p.(Arg79Ter), c.136C>T p.(Arg46Ter), and c.595C>T p.(Arg199Ter)) impair peroxisomal fission, leading to abnormal peroxisome morphology and distribution .

• Disruption of metabolic functions: Patient studies have revealed biochemical abnormalities associated with PEX11B mutations, including very low plasmalogens in some patients and mildly deranged very long chain fatty acid profiles in others . These metabolic disturbances likely contribute to the systemic manifestations of the disorder.

• Developmental impacts: Congenital cataracts appear to be a consistent feature in patients with PEX11B mutations, suggesting that PEX11B plays a critical role in lens development during embryogenesis . The variable presentation of other features like short stature, skeletal abnormalities, and dysmorphism indicates that PEX11B functions in multiple developmental pathways.

• Tissue-specific effects: The observation that some tissues are more affected than others in patients with PEX11B mutations suggests tissue-specific requirements for peroxisome division and metabolism, potentially related to differential expression of compensatory mechanisms or interaction partners .

Understanding these pathogenic mechanisms is crucial for developing potential therapeutic interventions and for accurate genetic counseling of affected families.

What is the relationship between PEX11B, SIRT1, and PPARγ in neural differentiation?

Recent research has uncovered a complex regulatory relationship between PEX11B, SIRT1, and PPARγ during neural differentiation of human embryonic stem cells (hESCs):

• PEX11B as a neural differentiation mediator: Loss of PEX11B expression through shRNA-mediated knockdown significantly decreases the expression of peroxisomal-related genes (ACOX1, PMP70, PEX1, PEX7) and reduces the formation of neural tube-like structures and neuronal markers .

• SIRT1's suppressive role: SIRT1 appears to have a suppressive effect on neural differentiation that is counteracted by PEX11B. Pharmacological inhibition of SIRT1 can partially rescue the neural differentiation defects caused by PEX11B knockdown, resulting in a relative increase in PEX11B expression and enhanced formation of neural tube-like structures .

• PPARγ as an essential mediator: The neuroprotective effects observed after SIRT1 inhibition are eliminated when PPARγ is inhibited, suggesting that PPARγ mediates the interaction between PEX11B and SIRT1. This indicates that PPARγ functions downstream of SIRT1 and upstream of PEX11B in this regulatory pathway .

• Integrated regulatory circuit: The data suggests a model where PPARγ positively regulates PEX11B expression, which in turn promotes neural differentiation, while SIRT1 acts as a negative regulator of this process. This creates a balanced regulatory system that can be fine-tuned during neural development .

This regulatory network provides new insights into the role of peroxisomal proteins in neural development and may have implications for understanding neurodevelopmental disorders associated with peroxisomal dysfunction.

What techniques are most effective for studying PEX11B-mediated peroxisome proliferation in cell culture systems?

Several complementary techniques have proven effective for investigating PEX11B function in cellular systems:

• Overexpression and knockdown studies:

  • Transfection of cells with PEX11B expression vectors induces substantial vesiculation of peroxisomes, providing a model to study the proliferation process

  • shRNA-mediated knockdown of PEX11B expression allows researchers to observe loss-of-function effects on peroxisome number and morphology

  • Both approaches can be combined with rescue experiments using wild-type or mutant PEX11B constructs to identify critical functional domains

• Live cell imaging techniques:

  • Fluorescent protein tagging of PEX11B and other peroxisomal markers enables real-time visualization of peroxisome dynamics

  • Time-lapse microscopy can capture the elongation, constriction, and fission events mediated by PEX11B

  • Super-resolution microscopy provides detailed insights into the distribution patterns of PEX11B on the peroxisomal membrane

• Biochemical and molecular assays:

  • Promoter-reporter assays using constructs like pGL3-basic vector with PEX11B promoter regions can identify regulatory elements controlling PEX11B expression

  • qPCR analysis of peroxisomal gene expression during different cellular states helps establish the temporal relationship between PEX11B expression and peroxisome proliferation

  • Co-immunoprecipitation studies can identify PEX11B interaction partners during different stages of peroxisome biogenesis

• In vitro reconstitution systems:

  • Proteo-liposome systems incorporating recombinant PEX11B proteins allow direct observation of membrane deformation effects under controlled conditions

  • These systems can be visualized using both confocal microscopy and electron microscopy to capture detailed morphological changes

Each method provides unique insights into PEX11B function, and combining multiple approaches yields the most comprehensive understanding of its role in peroxisome dynamics.

How can researchers effectively generate and validate recombinant human PEX11B protein for functional studies?

Generating high-quality recombinant human PEX11B requires specialized approaches due to its membrane protein nature:

• Expression system selection:

  • Bacterial systems (E. coli): May be suitable for producing specific domains (particularly soluble regions), but often struggle with full-length membrane proteins

  • Insect cell systems (Sf9, High Five): Generally more effective for expressing membrane proteins with proper folding

  • Mammalian expression systems: Provide the most native post-translational modifications and folding environment, but typically yield lower protein amounts

• Construct design considerations:

  • Include affinity tags (His, GST, FLAG) for purification, positioned to minimize interference with function

  • Consider removing transmembrane domains for improved solubility when studying specific domains

  • Include TEV or similar protease cleavage sites to remove tags after purification

  • Test different fusion partners to enhance solubility and expression

• Purification strategy:

  • Use detergent screening to identify optimal solubilization conditions

  • Implement multi-step purification including affinity chromatography, ion exchange, and size exclusion

  • Consider amphipol or nanodisc reconstitution for increased stability of the purified protein

• Validation approaches:

  • Conduct structural integrity assessment through circular dichroism spectroscopy

  • Verify oligomerization state using analytical ultracentrifugation or size exclusion chromatography

  • Perform functional validation in proteo-liposome systems to assess membrane deformation capabilities

  • Test binding to known interaction partners like DLP1 or Mff using surface plasmon resonance

• Activity assays:

  • Liposome tubulation assays to measure membrane deformation activity

  • GTPase stimulation assays to assess interaction with DLP1

  • Membrane binding assays to quantify lipid interaction properties

Successful recombinant PEX11B production requires careful optimization at each step, with particular attention to maintaining the protein's native structure and membrane-interaction capabilities.

What are the optimal approaches for analyzing PEX11B promoter regulation in different cell types?

Understanding PEX11B promoter regulation requires a multi-faceted approach:

• Promoter mapping and analysis:

  • Bioinformatic analysis to identify putative transcription factor binding sites, including SMAD2/3 binding sites associated with TGFβ signaling

  • Generation of promoter-reporter constructs with varying lengths of the PEX11B promoter region to identify key regulatory elements

  • Site-directed mutagenesis of specific binding sites (like the SMAD2/3 site) to confirm their functional significance

• Transcription factor binding studies:

  • Chromatin immunoprecipitation (ChIP) assays to verify direct binding of transcription factors such as SMAD2/3 to the PEX11B promoter in vivo

  • Electrophoretic mobility shift assays (EMSA) to confirm specific DNA-protein interactions in vitro

  • DNA pulldown assays coupled with mass spectrometry to identify novel transcription factors that bind to the PEX11B promoter

• Functional regulation studies:

  • Dual-luciferase reporter assays to quantify promoter activity under different conditions or treatments (e.g., TGFβ stimulation, serum stimulation, fatty acid supplementation)

  • Time-resolved mRNA expression analysis during peroxisome proliferation to establish temporal patterns of regulation

  • Treatment with pathway-specific agonists or antagonists (like PPARγ or SIRT1 modulators) to dissect regulatory mechanisms

• Cell-type specific considerations:

  • Compare PEX11B promoter activity across various cell types (fibroblasts, hepatocytes, neural cells) to identify tissue-specific regulatory mechanisms

  • Use cell-type specific transcriptomic data to correlate PEX11B expression with other genes and pathways

  • Consider epigenetic regulation through methylation analysis of the PEX11B promoter in different cell types

• Advanced techniques:

  • CRISPR-based approaches for endogenous promoter modification or transcription factor recruitment

  • Single-cell gene expression analysis to capture heterogeneity in PEX11B regulation within populations

  • 3D chromatin capture techniques to understand long-range interactions influencing PEX11B expression

These approaches should be tailored to the specific research question and cell system under investigation, with particular attention to the differential regulation observed between PEX11 isoforms.

How do PEX11B mutations manifest clinically and what is the genotype-phenotype correlation?

PEX11B mutations result in a spectrum of clinical manifestations with varying severity:

The expanding phenotypic spectrum of PEX11B mutations highlights the importance of considering this gene in patients with congenital cataracts, especially when accompanied by other features suggestive of peroxisomal dysfunction.

What experimental models best recapitulate human PEX11B-associated disorders for therapeutic development?

Several experimental models can be employed to study PEX11B-associated disorders:

• Cellular models:

  • Patient-derived fibroblasts provide a physiologically relevant system that maintains the genetic background of affected individuals

  • CRISPR/Cas9-engineered cell lines carrying specific PEX11B mutations enable controlled studies of mutation effects

  • iPSC (induced pluripotent stem cell) models derived from patient cells can be differentiated into relevant cell types like neurons or lens cells to study tissue-specific manifestations

  • Human embryonic stem cells with PEX11B knockdown have proven useful for studying neural differentiation defects

• Organoid models:

  • Eye organoids can help investigate the mechanisms of cataract formation in PEX11B deficiency

  • Brain organoids may reveal neurodevelopmental impacts of peroxisomal dysfunction

  • Liver organoids could provide insights into metabolic aspects of the disorder

• Animal models:

  • Mouse models with Pex11b knockout or knockin of human mutations can recapitulate systemic aspects of the disease

  • Zebrafish models offer advantages for high-throughput screening of potential therapeutic compounds

  • Drosophila models may be useful for genetic interaction studies due to their simpler genetic background

• Model selection considerations:

  • Match the model to the specific research question (e.g., molecular mechanisms vs. therapeutic screening)

  • Consider species-specific differences in peroxisome biology when interpreting results from animal models

  • Validate findings across multiple model systems to strengthen translational relevance

• Therapeutic screening approaches:

  • High-content imaging of peroxisome morphology in patient cells treated with compound libraries

  • Phenotypic rescue assays measuring restoration of peroxisomal functions

  • Gene therapy approaches using viral vectors to deliver functional PEX11B

The ideal approach often involves combining multiple models to build a comprehensive understanding of the disorder and to validate potential therapeutic strategies at different levels.

How might targeting PEX11B expression or activity be leveraged for therapeutic benefit in peroxisomal and neurodevelopmental disorders?

Emerging evidence suggests several potential therapeutic strategies targeting PEX11B:

• Gene therapy approaches:

  • Delivery of functional PEX11B using viral vectors (AAV, lentivirus) could potentially restore peroxisome division in patients with loss-of-function mutations

  • CRISPR-based gene editing could correct specific mutations in patient cells

  • Antisense oligonucleotides might be useful for modulating splicing in cases with splice-affecting mutations

• Pharmacological modulation of PEX11B expression:

  • PPARγ agonists might increase PEX11B expression, as suggested by the PPARγ-dependent regulation of PEX11B observed in neural differentiation studies

  • TGFβ pathway modulators could potentially enhance PEX11B expression through the SMAD2/3 binding site in its promoter

  • SIRT1 inhibitors have shown promise in counteracting the effects of PEX11B knockdown in neural differentiation models

• Metabolic bypass strategies:

  • Supplementation with plasmalogens or precursors might address the biochemical deficiencies observed in some patients with PEX11B mutations

  • Dietary modifications to reduce very long chain fatty acid accumulation could help normalize lipid profiles

• Targeted approaches for specific manifestations:

  • Early surgical intervention for congenital cataracts remains the standard of care

  • Neuroprotective agents might prevent or slow the progression of neurological manifestations

  • Growth hormone therapy could be considered for patients with short stature

• Combination therapies:

  • Multi-modal approaches targeting both peroxisome biogenesis and specific metabolic pathways may prove more effective than single interventions

  • Personalized approaches based on individual patient biochemical profiles could optimize therapeutic outcomes

Therapeutic development for PEX11B-associated disorders is still in its early stages, but the increasing understanding of PEX11B function and regulation provides promising avenues for future interventions.