PMP2 Human, His

What is PMP2 and what are its primary functions in human peripheral nerves?

PMP2 (also known as FABP8) is a small protein (~14 kDa) that functions as a structural component of peripheral nervous system myelin. It belongs to the family of cytoplasmic fatty acid-binding proteins and is thought to play crucial roles in:

• Stabilization of myelin membranes through membrane stacking

• Lipid transport in Schwann cells

• Potential binding of cholesterol within the myelin sheath

PMP2 is primarily expressed in peripheral nervous system myelin, though it is also present in smaller amounts in central nervous system myelin and has been detected in human astrocytes . As a myelin structural protein, it contributes to the integrity and function of the myelin sheath that insulates and protects nerve axons.

How does the structure of PMP2 relate to its biological function?

PMP2 contains a highly conserved lipocalin/cytosolic fatty acid binding domain that is critical for its functionality . Structurally, this domain creates a β-barrel that forms a hydrophobic pocket capable of accommodating fatty acids and potentially other lipid molecules. This structural arrangement facilitates:

• Binding and transport of fatty acids within Schwann cells

• Interaction with phospholipid membranes to assist in membrane stacking

• Stabilization of the compact myelin structure through protein-lipid interactions

What is the significance of using His-tagged PMP2 in research applications?

His-tagged human PMP2 provides several advantages for research purposes:

• Enables efficient purification using immobilized metal affinity chromatography

• Facilitates detection through anti-His antibodies in various assays

• Allows for controlled orientation during immobilization on surfaces for interaction studies

• Provides minimal interference with protein structure while enabling quantitative recovery

For structural studies, His-tagged PMP2 has been particularly valuable as it allows for the production of highly pure protein samples suitable for crystallization, which has been instrumental in determining the protein's three-dimensional structure through X-ray and neutron crystallography . The tag's small size minimizes its impact on protein folding and function, making it ideal for functional studies examining lipid binding and membrane interactions.

How do disease-causing mutations affect PMP2 structure and function at the molecular level?

Several PMP2 mutations have been identified in patients with Charcot-Marie-Tooth disease, including p.I43N, I50del, M114T, and V115A . These mutations affect PMP2 function through various mechanisms:

• Altered thermal stability: All known disease variants show reduced thermal stability compared to wild-type PMP2, suggesting increased susceptibility to denaturation under physiological conditions

• Impaired fatty acid binding: Disease variants demonstrate altered fatty acid binding properties, potentially affecting lipid transport function

• Preserved membrane stacking: Interestingly, vesicle aggregation assays show that membrane stacking characteristics remain largely unchanged despite the mutations

Crystal structures of these variants show only minor structural differences compared to wild-type PMP2, indicating that the pathological effects likely stem from altered dynamics or subtle changes in interaction surfaces rather than gross structural abnormalities .

What experimental approaches are most effective for studying PMP2-lipid interactions?

Several complementary methodologies have proven effective for investigating PMP2-lipid interactions:

• Fluorescent lipid binding assays: Using environmentally sensitive probes like 11-dansylaminoundecanoid acid (DAUDA) to quantify binding affinities and kinetics

• Vesicle aggregation assays: Assessing the ability of PMP2 to stack lipid membranes through turbidity measurements

• Time-lapse imaging of lipid bilayers: Direct visualization of PMP2-induced formation of double-membrane structures that may reflect its in vivo membrane stacking function

• Small-angle X-ray scattering (SAXS): Provides information about protein conformation in solution when bound to different lipids

• Molecular dynamics simulations: Complement experimental data by revealing the dynamic aspects of protein-lipid interactions

When working specifically with His-tagged PMP2, researchers can immobilize the protein on sensor chips via the His-tag for surface plasmon resonance studies, allowing real-time monitoring of lipid binding with minimal perturbation of the protein structure.

How do the dynamics of PMP2 contribute to its biological function and disease pathogenesis?

Recent studies combining neutron crystallography, X-ray diffraction at room temperature, and computer simulations have revealed important insights about PMP2 dynamics:

• PMP2 exhibits subtle but functionally significant internal dynamics that contribute to lipid binding and release

• Disease-causing mutations appear to alter these dynamic properties without drastically changing the static structure

• The neutron crystal structure of perdeuterated PMP2 refined from room temperature data provides a more physiologically relevant view of protein dynamics compared to cryocooled structures

These studies suggest that altered protein dynamics might be a key factor in disease pathogenesis, as they could affect:

• The kinetics of fatty acid binding and release

• Protein-membrane interactions essential for myelin stability

• Long-term protein stability in the myelin sheath

What expression systems are optimal for producing His-tagged human PMP2 for structural and functional studies?

For high-quality His-tagged human PMP2 production, consider these expression systems and methodologies:

• E. coli BL21(DE3): Most commonly used system for His-tagged PMP2 expression, typically using pET vectors with T7 promoter

• Perdeuteration: For neutron crystallography studies, expression in minimal media with D₂O as the solvent allows for production of perdeuterated protein, enabling more detailed structural analysis

• Expression conditions optimization:

  • Induction at lower temperatures (16-18°C) often improves solubility

  • IPTG concentration of 0.5-1.0 mM is typically optimal

  • Post-induction expression for 16-20 hours yields best results

Purification protocol typically involves:

• Immobilized metal affinity chromatography (Ni-NTA)

• Size exclusion chromatography for higher purity

• Optional tag removal using specific proteases if the tag might interfere with structural studies

For crystallization purposes, protein concentration of 10-15 mg/ml in a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl has been successfully used .

How can researchers effectively assess PMP2 mutations and their impact on myelin structure?

A multi-faceted approach combining in vitro, cellular, and animal model studies provides comprehensive understanding:

• In vitro biophysical characterization:

  • Circular dichroism spectroscopy to assess thermal stability changes

  • X-ray crystallography to determine structural alterations

  • Lipid binding assays to quantify changes in fatty acid affinity

• Cellular models:

  • Transfection of wild-type or mutant PMP2 into myelinating cells

  • Assessment of myelin formation through immunofluorescence microscopy

  • Live-cell imaging to track PMP2 localization and dynamics

• Animal models:

  • Transgenic mice expressing wild-type or mutant PMP2 provide valuable insights into in vivo effects

  • Electrophysiological measurements to assess nerve conduction velocities

  • Electron microscopy to visualize myelin ultrastructure

Research with transgenic mice has demonstrated that both overexpression of wild-type PMP2 and expression of mutant PMP2 (p.I43N) result in reduced motor nerve conduction velocities (MNCV) and demyelinating pathology, similar to findings in human CMT patients .

What methods are most effective for studying the membrane stacking function of PMP2?

Several complementary approaches can be employed to investigate PMP2's membrane stacking capabilities:

• Vesicle aggregation assays: Measuring turbidity changes when PMP2 is added to vesicle suspensions provides quantitative data on membrane stacking activity

• Time-lapse imaging of lipid bilayers: Direct visualization of double-membrane structures formed upon addition of PMP2 to model membranes

• Cryo-electron microscopy: Provides high-resolution images of PMP2-mediated membrane stacks

• Atomic force microscopy: Allows quantification of the forces involved in membrane stacking

• Fluorescence resonance energy transfer (FRET): Using differentially labeled membranes to quantify proximity induced by PMP2

Time-lapse imaging has been particularly informative, revealing that PMP2 can induce the formation of double-membrane structures that likely reflect its biological function in stacking two adjacent myelin membrane surfaces in vivo . This technique allows real-time monitoring of membrane interactions and can be particularly valuable for comparing wild-type and mutant proteins.

How are mutations in the PMP2 gene linked to Charcot-Marie-Tooth disease?

Mutations in the PMP2 gene have been identified as causes of autosomal dominant demyelinating Charcot-Marie-Tooth disease (CMT1), expanding our understanding of the genetic landscape of this disorder:

• The p.I43N mutation was identified in families with autosomal dominant demyelinating CMT neuropathy through whole exome sequencing

• Additional disease-associated mutations include I50del, M114T, and V115A

• CMT associated with PMP2 mutations has been classified as CMT1G

The pathogenic mechanisms include:

• Altered protein stability and dynamics

• Changes in fatty acid binding properties

• Potential impact on myelin membrane structure and stability

Interestingly, transgenic mouse models have shown that both overexpression of wild-type PMP2 and expression of mutant PMP2 can cause CMT1-like phenotypes, suggesting that proper PMP2 dosage is critical for normal myelin function . This mirrors findings with PMP22, another myelin protein associated with CMT.

What are the clinical and pathological characteristics of PMP2-associated Charcot-Marie-Tooth disease?

PMP2-associated CMT1 presents with distinct clinical and pathological features:

Clinical characteristics:

• Age of onset typically in the first or second decades of life

• Muscle atrophy beginning in the distal portions of the legs

• Progressive motor and sensory neuropathy

• Variable presence of hand tremors

• Pes cavus (high-arched feet) is common

• Functional disability ranges from mild to moderate

Imaging findings:

• MRI reveals predominant fatty replacement in the anterior and lateral compartment muscles of the lower leg

• Sequential pattern of muscle involvement correlates with disease duration

• Lateral compartment muscles (peroneus longus and brevis) show earliest and most severe involvement

• Posterior compartment muscles (soleus, gastrocnemius, tibialis posterior) remain relatively unaffected even in later disease stages

Pathological features:

• Sural nerve biopsy shows onion bulbs and degenerating fibers with various myelin abnormalities

• Demyelinating pattern with evidence of abnormal remyelination

• Electron microscopy in transgenic mouse models reveals shortened internodal lengths

These characteristics help distinguish PMP2-associated CMT from other genetic forms of the disease and can guide diagnostic workup.

How do animal models contribute to our understanding of PMP2-related neuropathies?

Animal models have provided crucial insights into the pathophysiology of PMP2-related neuropathies:

Transgenic mouse models:

• Mice expressing either wild-type or mutant (p.I43N) PMP2 exhibit abnormal motor function

• Electrophysiological studies show reduced motor nerve conduction velocities (MNCV), matching the human disease phenotype

• Electron microscopy reveals demyelinating fibers and shortened internodal lengths in both models

Key findings from animal studies:

• Both overexpression of wild-type PMP2 and expression of mutant PMP2 lead to similar CMT1 phenotypes

• This parallels findings with PMP22, where both duplication (CMT1A) and point mutations (CMT1E) cause disease

• The similarity between wild-type overexpression and mutant expression effects suggests that proper regulation of PMP2 levels is critical for normal myelin function

These animal models provide platforms for:

• Testing potential therapeutic approaches

• Longitudinal study of disease progression

• Detailed investigation of cellular and molecular mechanisms

• Preclinical evaluation of treatments targeting PMP2 function or expression

What are the future research directions for PMP2 in the context of myelin biology and neuropathies?

Several promising research avenues warrant further investigation:

• Structural dynamics: Further exploration of how protein dynamics, rather than static structure, contribute to PMP2 function and dysfunction

• Therapeutic targeting: Development of small molecules that could stabilize mutant PMP2 or modulate its lipid binding properties

• Gene therapy approaches: Investigating methods to normalize PMP2 expression levels in cases where overexpression contributes to pathology

• Interaction partners: Identification and characterization of proteins that interact with PMP2 in the myelin sheath

• Expanded mutation screening: Systematic screening for PMP2 mutations in undiagnosed CMT patients to better define the prevalence and spectrum of PMP2-related disease