Recombinant Human Protrudin (ZFYVE27)

What is the domain structure of Protrudin (ZFYVE27) and how does it relate to function?

Protrudin (ZFYVE27) contains several key functional domains that enable its role in membrane trafficking and neurite extension:

• N-terminal region: Contains a Rab11-binding domain (RBD11)

• Central region: Features three hydrophobic regions (HR1, HR2, and HR3), a FFAT motif, and a coiled-coil domain

• C-terminal region: Contains a FYVE domain that binds phosphoinositides

These structural elements enable Protrudin to function as a scaffold protein linking various cellular components. The HR3 region (amino acids 185-207) is particularly critical as it mediates self-interaction and oligomerization of Protrudin molecules . The FYVE domain binds to phosphatidylinositol 3-phosphate (PI3P), allowing interaction with endosomal membranes, while the FFAT motif mediates binding to VAP proteins on the endoplasmic reticulum .

How can Protrudin be characterized biochemically for research applications?

For biochemical characterization of Protrudin:

• Oligomerization assessment: Employ sucrose gradient centrifugation to determine the oligomeric state. Research shows that Protrudin primarily forms dimers/tetramers, which can be detected between fractions 4-8 of a 10-30% sucrose gradient .

• Membrane association analysis: Use subcellular fractionation followed by Triton X-114 membrane phase separation to determine Protrudin's membrane association properties. Results indicate Protrudin is a peripheral membrane protein with strong association to the ER membrane .

• Phosphoinositide binding: Conduct phosphoinositide binding assays to verify the functionality of the FYVE domain. Purified recombinant Protrudin should bind preferentially to PI3P-containing liposomes .

• Protein purity verification: Assess purity using SDS-PAGE, where recombinant human Protrudin typically appears as a 46-50 kDa band .

What expression systems are optimal for producing functional recombinant human Protrudin?

Several expression systems have been successfully used to produce functional recombinant Protrudin:

• Cell-free expression system: Yields functional transmembrane protein without the complications of cellular compartmentalization . This approach is particularly useful for structural studies as it avoids the challenges of membrane protein extraction.

• Mammalian expression systems: HEK293 or similar cell lines with vectors like pEF-BOS-2× or pEGFP-C1 are preferred for studying Protrudin in a native-like environment . This approach is advantageous when studying post-translational modifications and protein-protein interactions.

• Bacterial expression: GST-tagged fragments of Protrudin can be expressed in E. coli for domain-specific functional studies, particularly useful for protein-protein interaction studies .

When expressing full-length Protrudin, consider these methodological insights:

• Add glycerol (≥25%) to stabilize the protein in solution

• Avoid repeated freeze-thaw cycles which can promote unwanted higher-order oligomerization

• Express with appropriate tags (His, GST, or fluorescent protein) based on downstream applications

What are the most effective neuronal models for studying Protrudin's role in axon regeneration?

Based on published research, the following neuronal models have proven effective:

• Primary cortical neurons: Particularly useful for in vitro axotomy experiments. Neurons cultured for 10-14 days in vitro (DIV) develop mature axons that typically fail to regenerate after injury, making them ideal for testing Protrudin's regenerative effects .

• Retinal ganglion cells (RGCs): Both in vitro cultured RGCs and in vivo optic nerve injury models provide excellent systems to study CNS axon regeneration. The optic nerve crush model allows for clear visualization of regenerating axons labeled with tracers .

• NSC34 motor neuron-like cells: Useful for high-throughput screening of Protrudin mutants before moving to primary neurons .

Methodological considerations:

• For in vitro axotomy, use laser-based or physical transection at least 200μm from the cell body

• When overexpressing Protrudin, titrate expression levels as dose-dependent effects have been observed

• For in vivo studies, consider AAV-mediated delivery of Protrudin to adult neurons 2-3 weeks before injury

How does Protrudin's oligomerization state affect its function, and how can this be experimentally manipulated?

Protrudin's ability to form oligomers (primarily dimers/tetramers) is essential for its function in promoting neurite extensions. The HR3 domain (amino acids 185-207) is critical for this self-interaction .

Experimental approaches to study oligomerization:

• Yeast two-hybrid (Y2H) assays: Effective for mapping interaction domains. Direct-Y2H assays have confirmed that Protrudin interacts with itself through the HR3 region .

• Co-immunoprecipitation: Use differently tagged versions of Protrudin (e.g., E2-ZFYVE27 and c-Myc-ZFYVE27) to confirm self-interaction in mammalian cells .

• Dominant-negative approach: Express a mutant lacking the HR3 domain (ZFYVE27 ΔHR3) together with wild-type Protrudin to disrupt oligomerization. This approach has been shown to abolish neurite formation and cause cytoplasmic swelling .

Effects of manipulating oligomerization:

• Deletion of HR3 (ZFYVE27 ΔHR3) prevents neurite extension and causes swelling of cell soma

• Co-expression of ZFYVE27 ΔHR3 with wild-type Protrudin exerts a dominant-negative effect

• Point mutations in the HR3 region can alter oligomerization without completely abolishing it

What methodologies are recommended for studying Protrudin's interactions with spastin and other HSP-related proteins?

To investigate Protrudin's interactions with spastin and other hereditary spastic paraplegia (HSP)-related proteins:

• Co-immunoprecipitation: Use anti-spastin antibodies to pull down endogenous spastin and detect associated Protrudin, or vice versa. Studies show that the N-terminal domain of spastin (containing the MIT motif) mediates interaction with Protrudin .

• Domain mapping: Generate deletion constructs of both proteins to identify specific interaction domains. For example, the N-terminal domain of spastin is sufficient for Protrudin binding .

• Mutational analysis: Introduce specific mutations (e.g., G191V in Protrudin) to assess effects on protein-protein interactions. The G191V mutation in Protrudin reportedly reduces binding to spastin, potentially contributing to HSP pathology .

• Functional validation: Assess whether mutations that disrupt protein interactions also affect cellular phenotypes such as neurite extension or ER morphology .

Methodological insights:

• Different assays may yield different results; for example, Y2H may show complete loss of interaction while co-IP may show only partial reduction

• Overexpression systems can sometimes mask subtle interaction defects

• Consider using endogenous protein levels when possible to avoid artifacts

How does Protrudin regulate membrane trafficking to promote neurite extension?

Protrudin functions as a key scaffolding molecule at the interface of endosomes, ER, and the cytoskeleton to promote directional membrane transport essential for neurite extension:

• Rab11 regulation mechanism: Protrudin interacts with GDP-bound Rab11 through its N-terminal Rab11-binding domain (RBD11). This interaction helps direct recycling endosomes toward the growing neurite tip, providing membrane components necessary for extension .

• Kinesin-mediated transport: Protrudin facilitates the interaction between kinesin motor proteins (particularly KIF5A/B) and cargo molecules. It acts as an adapter protein linking KIF5A with VAPA, VAPB, SURF4, RAB11A, RAB11B, and RTN3, enabling their anterograde transport in neurons .

• ER-endosome contact sites: Protrudin localizes to ER tubules and forms contact sites with endosomes, facilitating the transfer of materials between these organelles. This is crucial for supplying growth-promoting molecules to the extending neurite .

Experimental evidence from recent studies:

• Overexpression of Protrudin increases anterograde transport of Rab11-positive recycling endosomes

• Constitutively active (phosphomimetic) Protrudin enhances the accumulation of endosomes at neurite tips

• Removal of Protrudin's kinesin-binding properties abolishes its regenerative effects

What techniques are most effective for visualizing Protrudin-mediated membrane trafficking in living neurons?

For real-time visualization of Protrudin-mediated trafficking in neurons:

• Live-cell imaging with dual-color labeling: Co-express fluorescently tagged Protrudin (e.g., GFP-ZFYVE27) with markers for different organelles (mCherry-Rab11 for recycling endosomes, ER-trackers for ER) . This approach allows simultaneous tracking of Protrudin and its cargo in living neurons.

• Fluorescence recovery after photobleaching (FRAP): Use FRAP to measure the kinetics of Protrudin movement within neuronal compartments. This technique can reveal directional bias in trafficking to growing neurites versus static regions .

• Microfluidic chambers for compartmentalized analysis: Culture neurons in microfluidic devices that separate cell bodies from axons, allowing selective manipulation and imaging of axonal Protrudin without contamination from somatic pools .

• Super-resolution microscopy: Techniques like STED or STORM provide nanoscale resolution of Protrudin localization at membrane contact sites between ER and endosomes .

Analytical considerations:

• Track individual vesicles to calculate velocity, directionality, and frequency of trafficking events

• Analyze the distribution of Protrudin along axons at different developmental stages

• Quantify colocalization with different organelle markers to determine Protrudin's compartmentalization

What are the optimal parameters for overexpressing Protrudin to promote axon regeneration in CNS neurons?

Research demonstrates that careful optimization of Protrudin expression is crucial for successful axon regeneration:

• Construct selection: Constitutively active (phosphomimetic) Protrudin consistently shows stronger regenerative effects than wild-type Protrudin. This form has phosphomimetic mutations at ERK phosphorylation sites, enhancing its activity .

• Expression levels: Protrudin's effect on axon regeneration is dose-dependent. Co-transfection with other constructs (like GFP) results in lower Protrudin expression and reduced regenerative effects . Aim for high expression levels while avoiding toxicity.

• Timing considerations: For in vitro studies, express Protrudin at least 24-48 hours before axotomy. For in vivo studies, allow 2-3 weeks for optimal expression before injury .

• Delivery methods:

  • For in vitro: Lipofection or nucleofection of plasmid DNA in mature neurons (>10 DIV)

  • For in vivo: AAV-mediated gene delivery to specific neuronal populations

Quantitative guidelines from research:

• In cortical neurons, active Protrudin expression results in ~44% of neurons extending axons beyond the injury site compared to ~8% in controls

• In the optic nerve, Protrudin overexpression enables axon regeneration up to 1.5mm beyond the crush site

How can researchers distinguish between Protrudin's effects on axon growth versus neuroprotection?

Differentiating Protrudin's regenerative versus neuroprotective effects requires carefully designed experiments:

• Temporal analysis:

  • For regeneration: Measure axon regrowth after confirmed axon degeneration

  • For neuroprotection: Assess neuronal survival rates and prevention of axon degeneration

• Selective outcome measures:

  • Regeneration metrics: Length of regenerating axons, growth cone morphology, branching patterns

  • Neuroprotection metrics: Cell viability assays, apoptotic markers (caspase-3), survival of injured neurons over time

• Combinatorial approaches: Use selective inhibitors of regeneration (e.g., growth cone collapsing factors) or neuroprotection (e.g., apoptosis inhibitors) to dissect the contribution of each mechanism.

• Domain-specific mutants: Create Protrudin variants with mutations in domains specifically affecting either regeneration or neuroprotection. For example:

  • FYVE domain mutants primarily affect regeneration by disrupting endosomal targeting

  • ER-localization mutants may predominantly impact neuroprotection through ER stress responses

Research findings to consider:

• Protrudin promotes both regeneration of injured axons and protection from degeneration

• In optic nerve injury models, Protrudin enhances RGC survival (~71% with Protrudin vs. ~44% in controls)

• Domain analysis shows that removing Protrudin's ER localization, kinesin-binding, or phosphoinositide-binding properties abrogates both regenerative and neuroprotective effects

What is the current understanding of Protrudin mutations in hereditary spastic paraplegia?

The relationship between Protrudin mutations and hereditary spastic paraplegia (HSP) remains controversial:

• Initial discovery: A G191V (G105V in ZFYVE27-c isoform) mutation in Protrudin was reported in a German family with autosomal dominant HSP, leading to the designation of ZFYVE27 as SPG33 .

• Subsequent investigation: Later studies identified this variant (rs35077384) as a polymorphism with the following allele frequencies in different populations:

  • 0.038 in APPLERA_GI:AGI_ASL population

  • 0.067 in HapMap-YRI population

  • 0.011 in HapMap-JPT population

  • 0.008 in HapMap-CEU population

• Functional impact: The G191V mutation reportedly:

  • Shifts a transmembrane domain by three amino acids

  • Reduces binding to spastin

  • Alters intracellular localization patterns

• Current consensus: The pathogenicity of this mutation is unclear, and some researchers suggest removing SPG33 from the list of HSP genes until incontrovertible pathogenic mutations are identified in other families .

Methodological considerations for researchers:

• Conduct thorough control screening when identifying potential pathogenic variants

• Perform functional validation of putative disease-causing mutations

• Consider the possibility that variants may act as modifiers for other HSP genes rather than causative mutations themselves

What experimental approaches are most promising for utilizing Protrudin to promote neural repair in disease models?

Several experimental approaches show promise for harnessing Protrudin's regenerative potential:

• Viral-mediated gene therapy: AAV vectors expressing constitutively active Protrudin can be delivered to injured CNS regions. This approach has shown success in optic nerve regeneration models .

• Cell-specific targeting: Use cell type-specific promoters to express Protrudin selectively in neurons affected by particular diseases or injuries. For example, in glaucoma models, target retinal ganglion cells specifically .

• Combinatorial approaches: Combine Protrudin overexpression with:

  • Manipulation of extrinsic inhibitory factors (e.g., PTEN deletion, SOCS3 inhibition)

  • Application of growth factors (e.g., BDNF, CNTF)

  • Modulation of the injury environment (e.g., inflammatory control)

• Small molecule screening: Identify compounds that enhance endogenous Protrudin activity or expression levels, which might avoid the challenges of gene therapy approaches.

Research findings supporting therapeutic potential:

• Constitutively active Protrudin enables robust CNS axon regeneration in the adult optic nerve

• Protrudin expression enhances neuronal survival after injury

• The effects are mediated through multiple cellular mechanisms including enhanced endosomal trafficking, ER dynamics, and axonal transport

Experimental design considerations:

• Include appropriate controls (GFP-only or wild-type Protrudin)

• Assess both functional recovery and anatomical regeneration

• Monitor for potential side effects of Protrudin overexpression

• Consider the timing of intervention relative to injury

How can researchers effectively manipulate the interaction between Protrudin and the endoplasmic reticulum for neurite extension studies?

To manipulate Protrudin-ER interactions for neurite extension research:

• Domain-specific mutations: Create variants with altered ER localization by:

  • Modifying the transmembrane domains or hairpin loop regions

  • Mutating the FFAT motif to disrupt VAP protein binding

  • Introducing targeted changes to the coiled-coil domain

• VAP protein manipulation: Knockdown or overexpress VAP-A/B proteins, which anchor Protrudin to the ER through FFAT domain interactions. Use shRNAs targeting specific VAP isoforms (like VAP-A-shRNA 1: 5′-GGTAGCACATTCGGATAAACC-3′) .

• ER morphology assessment: Quantify changes in ER network structure using:

  • ER-targeted fluorescent proteins (e.g., mCherry-KDEL)

  • Super-resolution microscopy to visualize ER tubule dynamics

  • Live-cell imaging to track ER movement into growing neurites

• Calcium signaling analysis: Since ER is a major calcium store, measure calcium dynamics during neurite extension to correlate ER positioning with local calcium signaling events.

Research findings on Protrudin-ER interactions:

• Protrudin functions in ER morphogenesis by regulating the sheet-to-tubule balance

• It contributes to both formation and stabilization of tubular ER networks

• Protrudin's hydrophobic hairpin domains generate high curvature in ER tubules

• Overexpression of active Protrudin facilitates ER accumulation in distal axons, correlating with enhanced regeneration

What emerging technologies might advance our understanding of Protrudin's role in axon regeneration?

Several cutting-edge technologies hold promise for deeper insights into Protrudin's functions:

• Optogenetic control of Protrudin activity: Develop light-sensitive Protrudin variants to achieve spatiotemporal control of its function in specific neuronal compartments. This would allow researchers to activate Protrudin selectively in growth cones or injury sites.

• CRISPR-Cas9 genome editing: Generate conditional knockout or knock-in models to study Protrudin function in specific neuronal populations or developmental stages. This approach could help resolve contradictory findings about Protrudin's role in disease.

• Proximity labeling technologies: Use BioID or APEX2 fused to Protrudin to identify proximal interacting proteins in different cellular compartments, revealing context-specific interaction networks.

• In vitro reconstitution systems: Develop membrane reconstitution assays to directly visualize and manipulate Protrudin-mediated membrane deformation and tubulation processes.

• Advanced imaging techniques:

  • Lattice light-sheet microscopy for high-speed, low-phototoxicity imaging of Protrudin dynamics

  • Expansion microscopy for nanoscale visualization of Protrudin localization

  • Correlative light and electron microscopy (CLEM) to link Protrudin's function to ultrastructural changes

• Single-cell transcriptomics: Compare gene expression profiles in Protrudin-overexpressing neurons to identify downstream pathways mediating regenerative effects.

Future research priorities:

• Clarify the controversial role of Protrudin in hereditary spastic paraplegia

• Identify the precise molecular mechanisms linking Protrudin to both regeneration and neuroprotection

• Develop therapeutic strategies targeting Protrudin for CNS injury and neurodegenerative diseases