Recombinant Pongo abelii Protrudin (ZFYVE27)

What is Pongo abelii Protrudin (ZFYVE27) and what are its structural characteristics?

Pongo abelii Protrudin (ZFYVE27) is a 411 amino acid protein from Sumatran orangutan that belongs to the FYVE family of proteins. It contains one FYVE-type zinc finger domain and several hydrophobic regions (HRs). Structurally, Protrudin is characterized by:

• A FYVE domain that mediates binding to phosphatidylinositol 3-phosphate

• Multiple hydrophobic regions, with HR3 (amino acids 185-207) being critical for self-interaction

• A zinc finger motif that plays a role in protein-protein interactions

Protrudin is primarily located in the endoplasmic reticulum membrane and functions in directional membrane trafficking .

What are the optimal storage and handling conditions for recombinant Pongo abelii Protrudin?

For optimal stability and activity of recombinant Pongo abelii Protrudin, the following storage and handling protocols are recommended:

These conditions are critical for maintaining protein integrity and function during experimental procedures .

How does ZFYVE27/Protrudin facilitate neurite extension and what experimental systems demonstrate this function?

Protrudin promotes neurite extension through several mechanisms:

• Directional membrane trafficking: Protrudin works with Rab11 to control directional membrane transport essential for neurite outgrowth .

• Interaction with cytoskeletal components: Protrudin interacts with spastin, a microtubule-severing protein, potentially coordinating cytoskeletal remodeling with membrane trafficking .

• Vesicular transport regulation: Protrudin serves as an adaptor connecting KIF5 motor proteins with vesicular cargoes during process formation .

Experimental systems demonstrating this function include:

• Overexpression studies showing that wild-type Protrudin induces neurite extensions in neuronal cells

• Loss-of-function experiments where cells expressing ZFYVE27 ΔHR3 (HR3 deletion mutant) fail to produce protrusions

• Co-localization studies showing Protrudin at the tips of neurite extensions

• Dominant negative effects observed when mutant and wild-type forms are co-expressed

What is the significance of Protrudin's oligomerization and how can it be experimentally demonstrated?

Protrudin's ability to form oligomers (primarily dimers and tetramers) is critical for its biological function. The significance includes:

• Oligomerization is necessary for Protrudin's ability to promote neurite extensions

• The HR3 region (amino acids 185-207) serves as the core interaction domain

• Deletion of HR3 results in a dominant-negative effect, preventing protrusion formation even when wild-type protein is present

This oligomerization can be experimentally demonstrated through multiple techniques:

These approaches provide complementary evidence for Protrudin's oligomerization and its functional significance .

What is the relationship between Protrudin and endoplasmic reticulum morphology?

Protrudin is an endoplasmic reticulum (ER)-anchored protein that plays a critical role in regulating ER morphology and function. Key aspects of this relationship include:

• ER network formation: Protrudin contributes to the formation and maintenance of the tubular ER network .

• Interaction with HSP-related proteins: Protrudin interacts with several hereditary spastic paraplegia (HSP)-related proteins that possess hairpin domains and regulate ER morphology .

• Contact site regulation: Protrudin may be involved in the formation or regulation of ER-plasma membrane or ER-endosome contact sites .

• Pathological implications: Mutant forms of Protrudin associated with HSP can form microaggregates that induce ER stress, potentially contributing to neurodegeneration .

Research has shown that alteration of Protrudin expression or function directly impacts ER structure, highlighting its importance in maintaining proper ER morphology which is crucial for neuronal function .

What is the molecular mechanism by which the HR3 domain mediates Protrudin oligomerization and function?

The HR3 domain (amino acids 185-207) serves as the core interaction region for Protrudin self-association. The molecular mechanism involves:

• Direct protein-protein interaction: The HR3 region directly mediates self-association, as demonstrated through deletion construct analysis in yeast two-hybrid systems. Constructs containing HR3 (ZFYVE27 150-250) efficiently interact with full-length ZFYVE27, while deletion of HR3 abrogates this interaction .

• Conformational requirements: HR3 likely adopts a specific conformation that enables proper oligomerization. This conformation may be influenced by the membrane association of Protrudin .

• Functional consequences: When HR3 is deleted:

  • Cells fail to produce protrusions and instead exhibit swelling of cell soma

  • The mutant protein exerts a dominant-negative effect on wild-type Protrudin

  • The directional membrane trafficking function is disrupted

• Potential interaction with other domains: While HR3 is the primary interaction domain, the N-terminal region (amino acids 1-184) shows weak binding affinity to full-length Protrudin, suggesting auxiliary interactions .

These findings indicate that HR3-mediated oligomerization creates a functional scaffold necessary for Protrudin's role in membrane trafficking and neurite extension .

How do mutations in ZFYVE27 contribute to hereditary spastic paraplegia (HSP) pathology?

The relationship between ZFYVE27 mutations and hereditary spastic paraplegia (HSP) involves complex mechanisms and some controversy:

• G191V mutation identification: A G191V missense mutation in ZFYVE27 was originally identified in a family with autosomal dominant HSP (designated SPG33). This mutation occurs within the critical HR3 domain (amino acids 185-207) that mediates Protrudin oligomerization .

• Functional consequences:

  • The mutation may disrupt Protrudin's ability to form proper oligomers

  • It potentially affects interaction with spastin (SPG4), the most common HSP-associated protein

  • It could impair directional membrane trafficking required for neurite extension

• Controversy: Some researchers have questioned whether ZFYVE27 should be classified as an HSP gene (SPG33). Martignoni et al. suggested that the G191V change might be in linkage disequilibrium with the real mutation or act as a modifier for another HSP gene .

• ER stress mechanisms: Mutant Protrudin may form microaggregates that induce ER stress, contributing to neurodegeneration. This connects to other HSP genes involved in ER morphology regulation .

Research continues to explore whether mutations directly cause HSP or contribute to pathology through interactions with other HSP proteins and pathways .

What techniques are most effective for studying Protrudin's interactions with binding partners and membrane structures?

Advanced research into Protrudin's interactions requires sophisticated methodological approaches:

For experimental validation of recombinant protein functionality, research shows that functional assays should include:

• Neurite extension assays in neuronal cell lines

• ER morphology analysis using confocal microscopy

• Dominant-negative suppression tests with mutant constructs

These approaches have been successfully employed to characterize Protrudin's interactions with spastin, atlastins, VAP proteins, and phosphoinositides .

What is the evolutionary conservation of Protrudin across species and what does this reveal about its function?

Protrudin (ZFYVE27) shows significant evolutionary conservation across species, providing insights into its fundamental biological roles:

• Sequence conservation: Comparative analysis between Pongo abelii (Sumatran orangutan) Protrudin and human Protrudin reveals high sequence similarity, particularly in functional domains like the FYVE domain and hydrophobic regions (HRs) .

• Functional domains: The HR3 region (amino acids 185-207), critical for oligomerization, shows strong conservation across primates, suggesting the fundamental importance of this domain for Protrudin function .

• Interacting partners: Protrudin's interaction with proteins like spastin, Rab11, and VAP is conserved across species, indicating preserved roles in membrane trafficking and ER organization .

• Species-specific variations: While core functions are preserved, species-specific variations may reflect adaptations in neuronal development and membrane trafficking processes.

This evolutionary conservation underscores Protrudin's fundamental roles in cellular processes, particularly in neurite extension and directional membrane trafficking, which are essential for proper neuronal development and function across species .

What are the best expression systems and purification strategies for producing functional recombinant Pongo abelii Protrudin?

For optimal expression and purification of functional recombinant Pongo abelii Protrudin, researchers should consider the following strategies:

• Expression systems comparison:

• Purification strategies:

  • Affinity purification using His-tag at the N-terminus has proven effective

  • For highest purity (>90%), SDS-PAGE-based quality control is essential

  • Purification under native conditions preserves functional oligomeric states

• Critical parameters:

  • Proper buffer composition (Tris-based with glycerol) maintains stability

  • Addition of protease inhibitors prevents degradation

  • Purification temperature (4°C) minimizes protein degradation

  • Proper tag selection (N-terminal vs. C-terminal) can impact functionality

Based on published research, E. coli-expressed His-tagged recombinant Pongo abelii Protrudin has been successfully used for functional studies, though mammalian expression may be preferable for certain applications .

How can researchers effectively analyze the oligomeric state of Protrudin in experimental systems?

Analyzing Protrudin's oligomeric state requires a multi-faceted approach:

• Sucrose gradient centrifugation protocol:

  • Prepare 5-20% sucrose gradients in appropriate buffer

  • Layer purified protein or cell lysate containing Protrudin on top

  • Centrifuge at 100,000 × g for 16-20 hours at 4°C

  • Collect fractions and analyze by immunoblotting

  • Include size standards to determine oligomeric state (dimer/tetramer)

• Cross-linking analysis:

  • Treat purified protein or intact cells with cross-linking reagents (e.g., DSS, formaldehyde)

  • Analyze by SDS-PAGE and immunoblotting

  • Compare migration patterns with molecular weight standards

  • Identify oligomeric species based on apparent molecular weight

• Native PAGE analysis:

  • Prepare samples without reducing agents or SDS

  • Run on gradient native gels (4-16%)

  • Compare migration patterns with native molecular weight standards

  • Confirm with immunoblotting or mass spectrometry

• Analytical ultracentrifugation:

  • Provides precise determination of molecular weight and oligomeric state

  • Can distinguish between different oligomeric forms in solution

  • Requires specialized equipment but offers high resolution

Previous research has utilized sucrose gradient centrifugation to demonstrate that Protrudin primarily forms dimer/tetramer complexes, with the HR3 domain being critical for this oligomerization .

What approaches are most effective for studying Protrudin's role in neurite extension and neurodegeneration models?

To effectively study Protrudin's role in neurite extension and neurodegeneration, researchers should consider these methodological approaches:

• Cellular models for neurite extension studies:

  • Primary neuronal cultures from cortex, hippocampus, or dorsal root ganglia

  • Neuronal cell lines (e.g., PC12, SH-SY5Y, Neuro2A) with differentiation protocols

  • iPSC-derived neurons from control and HSP patient samples

• Genetic manipulation strategies:

  • CRISPR/Cas9 for gene knockout or knock-in of specific mutations

  • shRNA or siRNA for transient knockdown

  • Overexpression of wild-type or mutant constructs

  • Domain-specific deletions (e.g., ΔHR3) for mechanistic studies

• Quantitative neurite analysis techniques:

  • High-content imaging with automated neurite tracing software

  • Live-cell imaging to track neurite dynamics over time

  • Specific markers for axons (Tau-1) and dendrites (MAP2)

  • Protrusion formation and length measurements in non-neuronal cells

• Molecular pathway analysis:

  • Proteomic approaches to identify interacting partners in different cellular compartments

  • Transcriptomic profiling to identify downstream effectors

  • Differential gene expression analysis between control and Protrudin-deficient cells

• Disease modeling approaches:

  • Transgenic mouse models expressing wild-type or mutant Protrudin

  • Patient-derived cells carrying HSP mutations

  • In vivo analysis of neuronal development and degeneration

Research has demonstrated that deletion of the HR3 domain causes dominant-negative effects on neurite extension, providing a valuable tool for studying Protrudin's function in neuronal development and potentially in neurodegeneration models .

What are the most promising approaches for translating basic Protrudin research into therapeutic strategies for hereditary spastic paraplegia?

Translational research on Protrudin presents several promising avenues for developing HSP therapeutics:

• Small molecule screening:

  • Compounds that enhance Protrudin function could compensate for partial loss of function

  • Molecules that stabilize Protrudin oligomerization may restore function of certain mutants

  • High-throughput screening using neurite extension as a functional readout

• Gene therapy approaches:

  • Delivery of wild-type ZFYVE27 to compensate for mutant protein

  • CRISPR/Cas9-based correction of specific mutations

  • Antisense oligonucleotides to modulate expression of mutant alleles

• Targeting interacting pathways:

  • Enhancing RAB11 function to bypass Protrudin defects in directional trafficking

  • Modulating ER stress responses to ameliorate consequences of mutant Protrudin

  • Enhancing spastin or other interacting proteins' function to compensate for Protrudin deficiency

• Biomarker development:

  • Identifying cellular or molecular signatures of Protrudin dysfunction

  • Developing assays to monitor disease progression and therapeutic response

  • Patient stratification based on molecular mechanisms

How can advanced imaging and 'omics techniques advance our understanding of Protrudin's function in neuronal development and degeneration?

Advanced technologies offer unprecedented opportunities to elucidate Protrudin's functions:

• Super-resolution microscopy applications:

  • Visualizing Protrudin localization at ER-endosome contact sites

  • Tracking dynamic interactions with binding partners during neurite extension

  • Monitoring changes in ER morphology with single-tubule resolution

  • Correlative light-electron microscopy to connect molecular localization with ultrastructure

• Spatially-resolved transcriptomics and proteomics:

  • Mapping region-specific changes in developing neurons with/without Protrudin

  • Identifying local translation events influenced by Protrudin activity

  • Analyzing compartment-specific proteomes in neurites vs. cell bodies

• Systems biology approaches:

  • Network analysis of Protrudin interactors across neuronal development stages

  • Integration of transcriptomic, proteomic, and phenotypic data

  • Computational modeling of membrane trafficking dynamics

• Single-cell analyses:

  • Examining cell-to-cell variability in Protrudin expression and function

  • Trajectory analysis during neuronal differentiation and maturation

  • Identification of cellular subpopulations particularly vulnerable to Protrudin dysfunction

These technologies can address fundamental questions such as how Protrudin coordinates with other HSP proteins, the spatiotemporal dynamics of its activity during neurite extension, and the molecular consequences of disease-associated mutations at cellular and subcellular levels .