PEX26 Human

What is PEX26 and what is its primary function in human cells?

PEX26 is a peroxisomal biogenesis factor that belongs to the peroxin-26 gene family. Its primary function is facilitating protein import into peroxisomes by anchoring PEX1 and PEX6 to peroxisome membranes, forming heteromeric AAA ATPase complexes essential for protein import . PEX26 is classified as a tail-anchored protein with C-terminal targeting information sufficient for correct sorting to the peroxisomal membrane . The gene encoding PEX26 is located on human chromosome 22q11.21 and has several known synonyms including PEX26PM1T, PEX26M1T, PBD7B, and PBD7A .

How does PEX26 reach the peroxisomal membrane?

The targeting mechanism of PEX26 to peroxisomes involves a specific pathway that differs from many other peroxisomal membrane proteins. Unlike proteins that follow a direct PEX19-dependent pathway, PEX26 first enters the endoplasmic reticulum (ER) in mammalian cells . This entry into the ER occurs independently of the GET/TRC40 machinery, which contrasts with the mechanism observed in yeast for the functional analog Pex15 .

Research has demonstrated that the C-terminal region of PEX26 contains sufficient targeting information for correct sorting to the peroxisomal membrane, functioning as a membrane peroxisomal targeting signal (mPTS) . After insertion into the ER membrane, PEX26 is believed to be trafficked to the peroxisomal membrane through a vesicular transport system. This indirect pathway highlights the complexity of peroxisomal membrane protein targeting and suggests an evolutionary connection between peroxisomes and the ER.

What are the clinical consequences of PEX26 mutations?

Mutations in the PEX26 gene cause peroxisome biogenesis disorders (PBDs) of complementation group 8 . These disorders arise from failures in protein import into the peroxisomal membrane or matrix. The clinical manifestations span a spectrum ranging from severe Zellweger syndrome (ZS) (OMIM# 615872) to less severe infantile Refsum disease (IRD) (OMIM# 614873) .

Patients with PEX26 mutations typically present with a constellation of clinical features including developmental delay, hypotonia, hepatic dysfunction, cholestasis, and neurological abnormalities. For instance, a specific mutation (c.347 T>A, p.(Leu116Gln)) in the PEX26 gene has been associated with cholestatic hepatopathy and developmental delay in patients from Dagestan . These patients developed symptoms in the first months of life, including severe hepatic dysfunction, jaundice, hepatosplenomegaly, and coagulopathy, alongside elevated levels of very-long-chain fatty acids (VLCFAs) in plasma .

The PBD group encompasses several disorders including Zellweger syndrome (ZWS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and classical rhizomelic chondrodysplasia punctata (RCDP) . The severity and specific manifestations depend on the nature of the PEX26 mutation and its impact on protein function.

What experimental approaches are most effective for studying PEX26 localization and trafficking?

Studying PEX26 localization and trafficking requires a combination of molecular biology, biochemistry, and advanced imaging techniques. Several methodological approaches have proven particularly valuable:

• Fluorescent protein tagging: Fusion of fluorescent proteins (e.g., GFP) to PEX26 enables real-time visualization of its localization and trafficking in living cells using confocal microscopy. This approach has been instrumental in tracking PEX26 movement from the ER to peroxisomes .

• Subcellular fractionation: Differential centrifugation techniques can separate cellular compartments, allowing detection of PEX26 in different fractions (peroxisomes, ER, cytosol) using western blotting with specific antibodies. This biochemical approach provides quantitative data on PEX26 distribution.

• Immunofluorescence microscopy: Using antibodies against PEX26 and markers for peroxisomes (e.g., PEX14) and the ER (e.g., calnexin) can determine the localization and trafficking route of PEX26. Co-localization studies have been key to establishing the ER-to-peroxisome pathway.

• Pulse-chase experiments: Metabolic labeling of newly synthesized PEX26 followed by chase periods can track its movement from the ER to peroxisomes over time, providing kinetic information about trafficking.

• Truncation and mutation analysis: Creating constructs with specific deletions or point mutations has demonstrated that C-terminal targeting information in PEX26 is sufficient for correct sorting to the peroxisomal membrane .

These methodologies, used in combination, have revealed that PEX26 follows an indirect pathway to peroxisomes via the ER, contrasting with the direct targeting of many other peroxisomal membrane proteins.

How do researchers detect and analyze PEX26 gene mutations in patient samples?

Several molecular genetic techniques are employed for detecting PEX26 gene mutations in patient samples:

• Next-Generation Sequencing (NGS) panels: Targeted sequencing of genes associated with peroxisomal disorders provides an efficient first-line approach. In clinical cases from Dagestan, panels targeting coding exons of 52-56 genes associated with inherited diseases with cholestasis, including PEX genes, were used successfully .

• Whole Exome Sequencing (WES): When targeted panels yield negative results or when the clinical presentation is atypical, WES examines all coding regions of the genome to identify PEX26 mutations.

• Sanger sequencing: This method confirms variants identified by NGS and enables familial testing. In the Dagestan cases, Sanger sequencing verified the c.347 T>A mutation in affected individuals and family members .

• PCR-RFLP (Restriction Fragment Length Polymorphism): This technique can screen specific known mutations in populations. The Dagestan study employed PCR-RFLP to test dried blood spot samples from 537 newborns for the presence of the c.347 T>A mutation .

For accurate interpretation of variants, bioinformatic algorithms such as PolyPhen-2, SIFT, PROVEAN, and MutationTaster predict the potential impact of missense variants . Population databases help determine the frequency of identified variants, which is crucial for assessing pathogenicity.

The comprehensive approach used in the Dagestan study—combining NGS, Sanger confirmation, bioinformatic prediction, and population screening—exemplifies the multifaceted strategy needed for thorough genetic analysis of PEX26 variants.

What biochemical assays are essential for detecting peroxisomal dysfunction in PEX26-deficient cells?

Several biochemical assays are critical for detecting and characterizing peroxisomal dysfunction in PEX26-deficient cells:

• VLCFA analysis: Gas chromatography–mass spectrometry (GC–MS) measures levels of very-long-chain fatty acids, particularly C26:0, and calculates ratios such as C26/C22 and C24/C22. In PEX26-deficient patients, C26:0 levels and the C26/C22 ratio are typically elevated . The Dagestan patients showed C26:0 levels of 13.2 mM/ml (normal range: 0.22–2.2 mM/ml) and a C26/C22 ratio of 0.149 (normal range: 0.009–0.018) .

• Phytanic acid analysis: Phytanic acid is metabolized in peroxisomes and accumulates in PBDs. Patients with the c.347 T>A PEX26 mutation exhibited phytanic acid levels of 59.42 mg/ml compared to the normal range of 0–3.11 mg/ml .

• Plasmalogen synthesis: Measurement of plasmalogens or their precursors using thin-layer chromatography or mass spectrometry identifies reduced plasmalogen levels, another indicator of peroxisomal dysfunction.

• Catalase latency test: This assay measures the accessibility of catalase to its substrate. In PEX26-deficient cells with impaired matrix protein import, catalase may be mislocalized to the cytosol rather than sequestered within intact peroxisomes.

• Immunofluorescence microscopy: Using antibodies against peroxisomal matrix proteins (e.g., catalase, thiolase) and membrane proteins (e.g., PEX14) assesses protein import and peroxisome morphology, providing visual evidence of peroxisomal defects.

These biochemical assays provide diagnostic information and can assess the severity of peroxisomal dysfunction associated with specific PEX26 mutations. The pattern of abnormalities helps distinguish PEX26 deficiency from other peroxisomal disorders and guides genetic testing.

How do researchers differentiate between direct and indirect pathways of PEX26 targeting to peroxisomes?

Differentiating between direct and indirect pathways of PEX26 targeting to peroxisomes requires specific experimental designs that can distinguish these routes:

• Brefeldin A treatment: This drug disrupts vesicular transport from the ER. If PEX26 trafficking to peroxisomes is inhibited by Brefeldin A, it suggests an indirect ER-dependent pathway, which has been observed in studies of PEX26 trafficking .

• Temperature blocks: Incubating cells at 15-16°C blocks exit from the ER, while 20°C blocks exit from the Golgi. These temperature blocks help delineate the trafficking pathway and have been used to demonstrate ER involvement in PEX26 targeting.

• Depletion of trafficking factors: siRNA-mediated knockdown or CRISPR-Cas9 knockout of factors involved in direct targeting (e.g., PEX19) or ER-to-peroxisome trafficking reveals which pathway PEX26 utilizes. Research has shown that PEX26 enters the ER independently of PEX19 .

• Heterologous expression systems: Expressing human PEX26 in yeast cells demonstrated that it can reach peroxisomes in these cells, suggesting conserved targeting information between species despite evolutionary divergence . Similarly, yeast Pex15 can target to peroxisomes in mammalian cells, further supporting the conservation of targeting mechanisms.

Research has established that in mammalian cells, PEX26 enters the ER independently of the GET/TRC40 machinery, which differs from the mechanism in yeast . This finding highlights both conserved and divergent aspects of peroxisomal protein import across species and demonstrates the importance of comparative studies in understanding fundamental targeting mechanisms.

What are the molecular consequences of pathogenic PEX26 variants at the cellular level?

Pathogenic variants in PEX26 trigger a cascade of cellular consequences that ultimately result in peroxisome dysfunction:

• Impaired peroxisome matrix protein import: Mutations in PEX26 disrupt its ability to anchor PEX1 and PEX6 to the peroxisomal membrane, compromising the recycling of the PEX5 receptor. This leads to defective import of peroxisomal matrix proteins containing PTS1 signals.

• Accumulation of metabolites: Due to impaired peroxisomal function, very-long-chain fatty acids (VLCFAs) and phytanic acid accumulate in cells and plasma. Patients with the c.347 T>A variant showed dramatically increased levels of C26:0 (13.2 mM/ml) and phytanic acid (59.42 mg/ml) .

• Altered peroxisome morphology: Depending on the severity of the mutation, peroxisomes may appear as empty "ghosts," reduced in number, or enlarged due to the import defect. These morphological changes can be visualized using electron microscopy or fluorescence microscopy with peroxisomal markers.

• Hepatic dysfunction: PEX26 mutations can cause severe liver abnormalities, including cholestasis, hepatomegaly, and coagulopathy . The liver dysfunction likely results from the accumulation of toxic metabolites that are normally processed by peroxisomes.

• Developmental impacts: The peroxisomal defects affect multiple organ systems during development, leading to neurological abnormalities, craniofacial dysmorphism, and growth retardation observed in patients with PEX26 mutations .

Understanding these molecular consequences provides insights into disease pathogenesis and potential therapeutic targets. The diverse effects underscore the essential role of peroxisomes in multiple metabolic pathways and developmental processes.

How can structure-function relationships in PEX26 be systematically investigated?

Investigating structure-function relationships in PEX26 involves several complementary approaches:

• Domain mapping: Creating truncation constructs and point mutations identifies functional domains. Research has established that the C-terminal domain of PEX26 contains targeting information sufficient for peroxisomal localization . Systematic mutation of specific residues, such as the leucine at position 116 that is affected in the Dagestan patients, provides insights into critical functional sites .

• Protein-protein interaction studies: Techniques including yeast two-hybrid, co-immunoprecipitation, and bimolecular fluorescence complementation map interaction interfaces between PEX26 and its binding partners, particularly PEX1 and PEX6.

• Cross-species complementation: Expressing PEX26 in yeast cells lacking Pex15 (its functional analog) has identified conserved functional regions despite limited sequence homology . This approach has demonstrated that human PEX26 can reach peroxisomes when expressed in yeast cells, highlighting evolutionarily conserved targeting mechanisms.

• Patient mutation analysis: Studying the effects of disease-causing mutations provides valuable information about critical functional regions. The c.347 T>A, p.(Leu116Gln) mutation affects a conserved leucine residue, suggesting this position is important for PEX26 function .

• Membrane topology studies: Protease protection assays and epitope insertion approaches determine which regions of PEX26 face the cytosol versus the peroxisomal lumen, providing insights into its functional organization within the membrane.

By integrating these approaches, researchers can develop a comprehensive understanding of how PEX26's structure relates to its functions in peroxisomal protein import and how specific mutations disrupt these functions to cause disease.

What diagnostic algorithm should be followed when suspecting PEX26-related disorders?

A comprehensive diagnostic algorithm for PEX26-related disorders should include:

• Clinical evaluation:

  • Assessment for characteristic features: developmental delay, hypotonia, hepatomegaly, dysmorphic features

  • Evaluation of liver function (transaminases, bilirubin, GGT)

  • Neurological examination and imaging

  • Ophthalmological examination

• First-line biochemical testing:

  • Plasma VLCFA analysis (elevated C26:0 and C26/C22 ratio)

  • Phytanic and pristanic acid levels

  • Erythrocyte plasmalogen levels

• Molecular genetic testing:

  • Targeted gene panel for peroxisomal disorders including PEX26 and other PEX genes

  • If negative, consider whole exome sequencing

  • Confirmation of variants by Sanger sequencing

  • Parental testing to confirm segregation

• Confirmatory biochemical tests:

  • Peroxisomal enzyme activities in fibroblasts

  • Immunofluorescence studies for peroxisomal protein localization

For infants presenting with cholestasis and hepatic dysfunction, biochemical screening for peroxisomal disorders should be included in the diagnostic workup, as illustrated by the cases from Dagestan where severe hepatic dysfunction was the initial presentation of PEX26 deficiency . These patients presented with symptoms similar to progressive familial intrahepatic cholestasis, highlighting the importance of considering peroxisomal disorders in the differential diagnosis of neonatal cholestasis.

Early diagnosis is crucial, as it allows for appropriate management and genetic counseling for families. The combination of biochemical and molecular testing provides the most accurate diagnostic approach for PEX26-related disorders.

How do genotype-phenotype correlations inform prognosis in PEX26-related disorders?

PEX26 mutations exhibit genotype-phenotype correlations across the Zellweger syndrome spectrum that inform prognosis:

• Severe phenotypes (Zellweger syndrome):

  • Typically associated with null mutations that completely abolish PEX26 function

  • Patients present with severe neurological abnormalities, characteristic facial features, and hepatomegaly

  • Prognosis is poor, with early mortality often in the first year of life

• Intermediate phenotypes (Neonatal Adrenoleukodystrophy):

  • Often caused by missense mutations that partially impair PEX26 function

  • Patients show developmental delay, hypotonia, hepatic dysfunction, and progressive neurological deterioration

  • Survival usually extends beyond infancy but with significant morbidity

• Milder phenotypes (Infantile Refsum Disease):

  • Associated with missense mutations that only mildly affect PEX26 function

  • Patients may survive into adolescence or adulthood with developmental delay, hearing and vision impairment

  • Long-term supportive care is often required, but life expectancy is significantly better than in more severe forms

• Variant-specific presentations:

  • The c.347 T>A, p.(Leu116Gln) variant has been associated with severe hepatic dysfunction and cholestasis as the initial presentation in the first months of life

  • These patients exhibited developmental delay but with distinctive predominance of liver involvement

Understanding these correlations helps clinicians provide more accurate prognostic information to families and develop appropriate management plans. The clinical variability observed with PEX26 mutations underscores the importance of comprehensive genetic and biochemical evaluation for each patient.

What therapeutic approaches are being investigated for PEX26-related disorders?

Several therapeutic approaches are being investigated for PEX26-related disorders, though most remain in experimental stages:

• Gene therapy approaches:

  • AAV-mediated gene delivery of functional PEX26 to affected tissues

  • CRISPR-Cas9 gene editing for correction of specific mutations

  • mRNA therapeutics for temporary expression of functional protein

• Small molecule interventions:

  • Read-through compounds for nonsense mutations

  • Chaperone therapies for misfolding mutations

  • Substrate reduction therapy to decrease accumulation of toxic metabolites

• Dietary interventions:

  • VLCFA restriction to limit dietary sources of very-long-chain fatty acids

  • DHA supplementation to provide essential fatty acids

  • Lorenzo's oil to help normalize VLCFA levels

• Symptomatic treatment:

  • For hepatic manifestations: ursodeoxycholic acid for cholestasis, vitamin K for coagulopathy

  • For neurological manifestations: anticonvulsants, physical therapy, occupational therapy

  • Supportive care addressing specific organ system involvement

• Emerging approaches:

  • Cell-based therapies including hepatocyte transplantation for liver-predominant disease

  • Exosome-based delivery of functional peroxisomal enzymes

  • Metabolic bypass strategies to reduce toxic intermediate accumulation

For patients with predominant liver involvement, as seen in the Dagestan cases with the c.347 T>A mutation , liver-directed therapies might be particularly relevant. Early intervention is essential, as many manifestations of peroxisomal disorders are developmental and potentially irreversible once established.

While curative therapies remain elusive, management focuses on supportive care and symptom mitigation. Advances in understanding PEX26 biology and the development of improved model systems offer hope for more effective treatments in the future.

What key unsolved questions remain regarding PEX26 biology?

Several critical questions about PEX26 function and regulation remain unanswered:

• Structural insights:

  • What is the three-dimensional structure of PEX26, and how does it interact with PEX1 and PEX6 at the molecular level?

  • How does the transmembrane domain of PEX26 anchor it to the peroxisomal membrane?

  • What structural features determine its initial targeting to the ER rather than directly to peroxisomes?

• Trafficking mechanisms:

  • What are the specific factors that mediate PEX26 insertion into the ER membrane?

  • How is PEX26 transported from the ER to peroxisomes, and what vesicular transport machinery is involved?

  • Why does PEX26 follow an indirect pathway via the ER rather than direct targeting to peroxisomes?

• Tissue-specific roles:

  • Why do some PEX26 mutations primarily affect liver function while others have more pronounced neurological effects?

  • Are there tissue-specific interaction partners or regulatory mechanisms?

  • How does PEX26 function differ during development versus in adult tissues?

• Evolutionary considerations:

  • Despite not being direct homologs, why can human PEX26 function in yeast and vice versa?

  • What evolutionary pressures have shaped the divergence of peroxisomal targeting mechanisms across species?

• Therapeutic targeting:

  • Can PEX26 function be rescued by small molecules in cells with specific mutations?

  • Are there bypass pathways that could compensate for PEX26 deficiency?

Addressing these questions will require integrating structural biology, cell biology, biochemistry, and systems biology approaches to fully understand PEX26's role in peroxisome biogenesis and disease pathogenesis.

How might emerging technologies advance PEX26 research?

Emerging technologies offer promising avenues to overcome current limitations in PEX26 research:

• Cryo-electron microscopy (Cryo-EM):

  • Determining the structure of the PEX1-PEX6-PEX26 complex at high resolution

  • Visualizing conformational changes during the ATPase cycle

  • Examining how mutations affect complex formation and function

• CRISPR-based technologies:

  • Genome-wide screens to identify factors affecting PEX26 targeting and function

  • Base editing to systematically assess the effects of specific amino acid changes

  • Creation of isogenic cell lines with disease-relevant mutations

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize PEX26 trafficking with nanometer precision

  • Live-cell imaging with single-molecule resolution to track PEX26 movement in real-time

  • Correlative light and electron microscopy to link protein localization with ultrastructural context

• Organoid and stem cell models:

  • Patient-derived organoids to study tissue-specific effects of PEX26 mutations

  • Brain and liver organoids to investigate organ-specific manifestations

  • Differentiation of iPSCs to study PEX26 function during development

• Systems biology approaches:

  • Multi-omics integration to understand the broader impact of PEX26 deficiency

  • Metabolic flux analysis to quantify changes in peroxisomal metabolism

  • Network modeling to identify potential compensatory pathways

These technologies, applied individually or in combination, have the potential to resolve long-standing questions about PEX26 biology and accelerate the development of therapeutic strategies for patients with PEX26-related disorders.