Recombinant Danio rerio Vacuolar protein sorting-associated protein 29 (vps29)

What is the functional role of VPS29 in the retromer complex of Danio rerio?

VPS29 functions as a key regulatory subunit of the retromer complex, which also includes VPS35 and VPS26. The retromer mediates endosomal protein sorting and recycling of transmembrane proteins from endosomes to the trans-Golgi network or plasma membrane. Based on studies in other model organisms, zebrafish VPS29 likely plays a crucial role in retromer localization and function, particularly in the nervous system. VPS29 appears to regulate retromer recruitment and release from endosomal membranes through interactions with regulatory proteins such as TBC1D5, a Rab7 GTPase-activating protein. This regulation follows a two-step mechanism: first, retromer is recruited to endosomal membranes via Rab7-GTP, and subsequently, VPS29 engages TBC1D5, which activates Rab7 GTP hydrolysis, facilitating retromer release from the endosome .

How conserved is VPS29 across species, and what does this suggest about zebrafish models?

VPS29 shows remarkable evolutionary conservation across species. Studies in Drosophila have demonstrated that human VPS29 can functionally substitute for fly VPS29, suggesting high structural and functional conservation . This conservation extends to zebrafish VPS29, which likely maintains the key structural features necessary for retromer function and interactions with regulatory proteins. The conservation is particularly evident in the hydrophobic pocket on VPS29 that interacts with the Pro-Leu motifs found in binding partners such as TBC1D5, VARP, and RidL . This high degree of conservation makes zebrafish an excellent model organism for studying VPS29 function with translational relevance to human biology and disease.

What expression and purification methods are most effective for recombinant Danio rerio VPS29?

For effective expression and purification of recombinant Danio rerio VPS29:

Expression System:

• Bacterial expression using E. coli BL21(DE3) with pET-based vectors

• Induction with IPTG at lower temperatures (16-18°C overnight) to enhance soluble protein yield

• Consideration of codon optimization for zebrafish genes expressed in bacterial systems

Purification Strategy:

• Initial affinity chromatography using His6, GST, or MBP tags

• Ion exchange chromatography as an intermediate purification step

• Size exclusion chromatography as a final polishing step to obtain monomeric protein

• Inclusion of reducing agents (DTT or TCEP) to maintain protein stability

• Buffer optimization (typically 20-50 mM Tris or HEPES, pH 7.5-8.0, 150-300 mM NaCl)

This approach typically yields highly pure, properly folded VPS29 suitable for structural and functional studies, as demonstrated in similar purification schemes for VPS29 from other species .

What phenotypes might be observed in zebrafish with VPS29 knockdown or knockout?

Based on studies in Drosophila and other model systems, zebrafish with VPS29 knockdown or knockout might exhibit:

Viability and Development:

• Unlike VPS35 or VPS26 knockouts which are often lethal, VPS29-deficient zebrafish may be viable but show reduced survival rates

• Possible developmental delays or subtle morphological abnormalities

Neurological Phenotypes:

• Progressive locomotor dysfunction with aging

• Synaptic transmission defects, particularly affecting sustained activity

• Retinal function abnormalities, including impaired photoreceptor function and synaptic transmission

Cellular Pathology:

• Mislocalization of other retromer components (especially VPS35) in neurons

• Accumulation of aberrant endolysosomal structures

• Increased numbers of multivesicular bodies and autophagic vacuoles

• Enlarged lysosomes with granular, electron-dense material

Drosophila studies show that VPS29 loss causes milder phenotypes than VPS35 or VPS26 deficiency, suggesting a regulatory rather than structural role in retromer function .

How can researchers verify the functional activity of purified recombinant zebrafish VPS29?

To verify functional activity of recombinant zebrafish VPS29:

Binding Assays:

• Pull-down assays with other retromer components (VPS35 and VPS26)

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify binding affinities

• Analytical size exclusion chromatography to verify complex formation

Protein-Protein Interaction Analysis:

• Binding assays with TBC1D5 fragments containing the VPS29-binding region

• Competition assays with peptides containing Pro-Leu motifs that mimic natural binding partners

• Co-crystallization trials with binding partners to verify structural integrity of interaction surfaces

Functional Assays:

• In vitro Rab7 GTPase activity assays to assess proper regulation of TBC1D5 activity

• Rescue experiments in VPS29-depleted zebrafish cells or tissues

• Cell-based cargo trafficking assays using fluorescently tagged retromer cargo proteins

These approaches provide complementary evidence for proper folding and function of the recombinant protein.

How might researchers characterize the interaction between zebrafish VPS29 and TBC1D5?

The interaction between zebrafish VPS29 and TBC1D5 can be characterized through:

Binding Interface Mapping:

• Mutagenesis of the conserved hydrophobic pocket on VPS29 that likely binds the Pro-Leu motif of TBC1D5

• Peptide array screening using TBC1D5-derived peptides to identify minimal binding motifs

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

Structural Analysis:

• X-ray crystallography of zebrafish VPS29 in complex with TBC1D5 peptides

• NMR studies to analyze conformational changes upon binding

• Molecular dynamics simulations to understand the dynamics of the interaction

Functional Characterization:

• In vitro Rab7 GTPase activity assays with purified components

• FRET-based biosensors to monitor VPS29-TBC1D5 interactions in zebrafish cells

• Rab7 activity probes to assess how mutations in the VPS29-TBC1D5 interface affect Rab7 GTPase cycling

These studies would build upon the known structural data showing that VPS29 contains a highly conserved pocket that binds Pro-Leu motifs found in TBC1D5, VARP, and the bacterial protein RidL .

What strategies can be employed to study the impact of VPS29 on retromer localization in zebrafish neurons?

To study VPS29's impact on retromer localization in zebrafish neurons:

Imaging Approaches:

• Generate transgenic zebrafish expressing fluorescently tagged VPS29, VPS35, and Rab7

• Employ confocal or super-resolution microscopy to visualize subcellular localization

• Use FRAP (Fluorescence Recovery After Photobleaching) to measure the kinetics of retromer association with endosomes

• Implement live imaging in zebrafish neurons to track retromer dynamics in real-time

Genetic Manipulation:

• Create VPS29 knockout zebrafish using CRISPR/Cas9

• Develop VPS29 mutants that specifically disrupt TBC1D5 binding without affecting retromer assembly

• Utilize conditional knockout systems to study temporal requirements for VPS29

Quantitative Analysis:

• Measure the ratio of neuropil to soma localization of other retromer components in the presence or absence of VPS29

• Track the formation of aberrant VPS35-positive structures in VPS29-deficient neurons

• Correlate retromer localization with functional readouts such as cargo trafficking

In Drosophila, VPS29 loss causes VPS35 to shift from neuropil to soma, forming large perinuclear puncta, suggesting that similar mislocalization might occur in zebrafish neurons .

How can researchers distinguish between direct effects of VPS29 manipulation versus secondary effects due to retromer dysfunction?

Distinguishing direct from secondary effects of VPS29 manipulation requires:

Structure-Function Approaches:

• Generate a panel of VPS29 mutants affecting different functional domains

• Compare phenotypic signatures across mutants affecting specific interactions

• Correlate molecular defects with cellular and organismal phenotypes

Temporal Analysis:

• Implement acute versus chronic VPS29 depletion strategies

• Establish a timeline of phenotypic changes following VPS29 manipulation

• Early-occurring phenotypes are more likely to represent direct effects

Comparative Studies:

• Compare phenotypes between VPS29, VPS35, and VPS26 manipulations

• Effects unique to VPS29 manipulation likely represent direct consequences

• Shared effects across all retromer components suggest secondary retromer dysfunction

Rescue Experiments:

• Perform rescue experiments with:

  • Wild-type VPS29

  • VPS29 mutants affecting specific interactions

  • VPS35 overexpression, which has been shown to partially rescue some VPS29 loss-of-function phenotypes

  • Downstream effectors or pathways

This approach leverages the observation that VPS29 appears to have a more regulatory role in retromer function, as evidenced by the milder phenotypes observed in VPS29 mutants compared to other retromer components .

What crystallization strategies would be most appropriate for structural studies of zebrafish VPS29?

For successful crystallization of zebrafish VPS29:

Protein Preparation:

• Express with a cleavable affinity tag and remove after initial purification

• Ensure >95% purity and monodispersity by size exclusion chromatography

• Optimize protein stability through buffer screening (e.g., Thermofluor assays)

• Consider methylation of surface lysines to promote crystal contacts

Crystallization Approaches:

• Screen multiple crystallization conditions using commercial sparse matrix screens

• Explore co-crystallization with binding partners such as:

  • VPS35 fragments

  • TBC1D5 peptides containing the Pro-Leu motif

  • Synthetic macrocyclic peptides that have been shown to bind VPS29 with high affinity

• Try both vapor diffusion and lipid cubic phase methods

Optimization Strategies:

• Fine-tune promising conditions by varying pH, precipitant concentration, and protein concentration

• Implement seeding techniques to improve crystal quality

• Consider surface entropy reduction mutations if initial crystallization attempts fail

• Use additives that have proven successful for other retromer component structures

Data Collection Considerations:

• Optimize cryoprotection to minimize background scatter

• Consider room-temperature data collection if cryoprotection proves challenging

• Collect high-resolution data at synchrotron sources with appropriate wavelength selection

The crystal structures of VPS29 from other species have provided valuable insights into binding interfaces , and similar approaches would be applicable to zebrafish VPS29.

How might synthetic macrocyclic peptides be utilized to study VPS29 function in zebrafish models?

Synthetic macrocyclic peptides offer powerful tools for studying VPS29 function in zebrafish:

Inhibitor Development:

• Design macrocyclic peptides based on those identified to bind VPS29 with high affinity (0.2-850 nM)

• Optimize peptides to specifically target the conserved binding pocket on VPS29

• Develop cell-permeable variants through chemical modifications

Functional Studies:

• Use peptides as competitive inhibitors of TBC1D5, VARP, and other binding partners

• Compare effects of different peptides targeting distinct interfaces on VPS29

• Develop control peptides with mutations in key binding residues

In Vivo Applications:

• Microinject peptides into zebrafish embryos to assess developmental effects

• Create transgenic zebrafish expressing peptide inhibitors under inducible promoters

• Develop methods for cell-type specific expression of inhibitory peptides

Therapeutic Exploration:

• Screen for peptides that can rescue defects in retromer function

• Identify peptides like RT-L4 that can act as molecular chaperones to stabilize retromer assembly

• Test peptides for ability to correct trafficking defects in disease models

These approaches build on research showing that macrocyclic peptides binding VPS29 can either inhibit interactions with accessory proteins or enhance retromer stability, depending on their binding site .

What approaches can identify novel interacting partners of zebrafish VPS29?

To identify novel VPS29 interacting partners:

Affinity Purification-Mass Spectrometry:

• Express tagged VPS29 in zebrafish cells or tissues

• Perform pull-downs under varying conditions (salt concentration, detergent, nucleotides)

• Use quantitative proteomics to distinguish specific from non-specific interactions

• Compare interactomes from different developmental stages or tissues

Proximity Labeling:

• Generate BioID or APEX2 fusions with zebrafish VPS29

• Express in relevant cell types or transgenic zebrafish

• Identify proximal proteins through biotinylation and streptavidin purification

• Compare proximity interactomes with direct binding partners

Peptide Screening:

• Screen synthetic peptide libraries for VPS29 binding

• Identify novel motifs that interact with the conserved pocket on VPS29

• Use bioinformatics to identify proteins containing these motifs

Crosslinking Mass Spectrometry:

• Apply chemical crosslinkers to purified retromer complexes or cellular extracts

• Identify crosslinked peptides through specialized mass spectrometry approaches

• Generate structural models of interaction interfaces

These approaches may reveal tissue-specific or context-dependent interactors beyond the known partners like TBC1D5 and VARP .

How can researchers develop zebrafish models to study the role of VPS29 in neurodegenerative conditions?

To develop zebrafish models for studying VPS29 in neurodegeneration:

Genetic Models:

• Generate constitutive or conditional VPS29 knockout zebrafish

• Create knock-in models expressing VPS29 variants associated with human disease

• Develop transgenic lines with fluorescently tagged VPS29 to monitor localization

Aging Studies:

• Characterize progressive phenotypes in VPS29-deficient zebrafish:

  • Locomotor function

  • Synaptic transmission

  • Neuronal ultrastructure

  • Protein aggregation

Disease-Relevant Readouts:

• Monitor endolysosomal morphology and function using fluorescent reporters

• Assess autophagy flux in neurons with VPS29 manipulation

• Examine protein aggregation of disease-relevant proteins (e.g., α-synuclein, tau)

• Evaluate synaptic degeneration using electrophysiology and imaging

Compound Testing:

• Screen for small molecules or peptides that rescue VPS29-deficient phenotypes

• Test whether VPS29 overexpression or stabilization can ameliorate neurodegeneration

• Evaluate compounds that modulate retromer function in the context of VPS29 manipulation

These approaches build on observations from Drosophila showing that VPS29 is required for maintenance of nervous system function during aging, with VPS29 mutants exhibiting progressive locomotor dysfunction and synaptic transmission defects .

What experimental designs can evaluate the role of VPS29 in regulating Rab7 activity in zebrafish?

To evaluate VPS29's role in regulating Rab7 activity:

Biochemical Assays:

• Purify zebrafish Rab7, VPS29, and TBC1D5

• Perform in vitro GTPase assays with various combinations of components

• Measure Rab7-GTP levels using GST-RILP pull-downs from zebrafish tissues

Imaging Approaches:

• Generate transgenic zebrafish expressing FRET-based Rab7 activity sensors

• Compare Rab7 activity patterns in wild-type versus VPS29-deficient neurons

• Perform live imaging to track spatiotemporal dynamics of Rab7 activation

Genetic Interactions:

• Test whether reducing Rab7 levels can suppress VPS29 loss-of-function phenotypes

• Evaluate the effects of constitutively active or dominant negative Rab7 in VPS29 mutants

• Examine whether TBC1D5 overexpression can rescue VPS29 deficiency

Ultrastructural Analysis:

• Use electron microscopy to characterize endolysosomal compartments in VPS29 mutants

• Correlate ultrastructural changes with alterations in Rab7 activity

• Implement correlative light and electron microscopy to link functional readouts with ultrastructure

These approaches build on the finding in Drosophila that Rab7 becomes hyperactivated and mislocalized in the absence of VPS29, and that reduction of Rab7 or overexpression of TBC1D5 can suppress VPS29 loss-of-function phenotypes .