VIPAS39 Antibody

What is VIPAS39 and why is it important in cellular research?

VIPAS39 (also known as C14orf133, VIPAR, or SPE-39) is a binding protein to Vps33B, one of the subunits in the mammalian HOPS (HOmotypic fusion and Protein Sorting) complex. It functions primarily in endosomal maturation or fusion processes . VIPAS39 is involved in endosome to lysosome transport and intracellular protein transport, and acts upstream of or within collagen metabolic processes and peptidyl-lysine hydroxylation .

The protein is particularly significant in research because:

• It plays a crucial role in the apical recycling pathway and maintenance of apical-basolateral polarity in epithelial cells

• Mutations in VIPAS39 cause arthrogryposis-renal dysfunction-cholestasis syndrome type 2 (ARCS2), a rare autosomal recessive multisystem disorder

• It provides insights into fundamental endosome traffic processes unique to metazoans

• It acts as a model for understanding protein-protein interactions in the endosomal sorting machinery

VIPAS39 is located in the Golgi apparatus and endosomes, and it is part of endosome and vesicle tethering complexes . The study of VIPAS39 offers valuable insights into basic cellular processes and disease mechanisms.

How do VIPAS39 and Vps33B interact, and what are the functional implications?

The interaction between VIPAS39 and Vps33B has been extensively characterized through multiple experimental approaches:

• Yeast two-hybrid (Y2H) analyses demonstrate that VIPAS39 robustly binds to Vps33b but not to other HOPS components like Vps11 or Vps18 . This suggests VIPAS39 associates with the class C core complex as a Vps33b-interacting protein rather than as a Vps16-like molecule .

• Co-immunoprecipitation studies confirm this interaction in mammalian cells, with wild-type Vps33b consistently co-immunoprecipitating with VIPAS39 .

• The interaction can be affected by certain mutations. For example:

  • The ARC mutation L30P in Vps33b disrupts VIPAS39-Vps33b association

  • The G249V mutation (a carnation-like mutation introduced into human Vps33b) prevents interaction

  • Other mutations like D234H, S243F (ARC mutations), and D252E (buff-like mutation) do not significantly affect binding

Functionally, the VIPAS39-Vps33B complex appears critical for:

• Endosomal maturation and/or fusion processes

• Proper trafficking of specific cargo proteins

• Maintenance of apical-basolateral polarity in epithelial cells

• Development and function of multiple organ systems, as evidenced by the multisystem nature of ARC syndrome

Interestingly, the VIPAS39-Vps33B interaction is conserved across species, from C. elegans to humans, suggesting its fundamental importance in cellular biology .

What are the optimal conditions for using VIPAS39 antibodies in Western blot experiments?

For Western blot (WB) experiments using VIPAS39 antibodies, the following optimized conditions are recommended based on validated commercial antibodies:

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results, as sample-dependent variations may occur . The antibody has demonstrated reactivity with both human and mouse samples .

When designing Western blot experiments to study VIPAS39 mutations or interactions, consider:

• Including both wild-type and mutant controls

• Using cross-linking agents like DSP to stabilize potentially weak protein-protein interactions

• Probing for known interaction partners (particularly Vps33B) on the same blot

• Validating results with multiple antibodies targeting different epitopes of VIPAS39

For particularly challenging samples or when studying low-abundance interactions, consider concentrating the protein by immunoprecipitation before Western blot analysis.

How can VIPAS39 antibodies be effectively used in immunofluorescence studies?

For effective immunofluorescence studies using VIPAS39 antibodies, implement the following protocol:

• Cell/Tissue Preparation:

  • For cultured cells: HeLa, HEK-293, and HepG2 cells have been validated for VIPAS39 antibody staining

  • For tissue sections: Liver tissue shows strong VIPAS39 expression and has been successfully used for immunohistochemistry

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • For tissue sections: Use TE buffer pH 9.0 for antigen retrieval (citrate buffer pH 6.0 is an alternative)

• Antibody Application:

  • Blocking: 5% BSA or normal serum in PBS for 30-60 minutes

  • Primary antibody: Apply VIPAS39 antibody at 1:10-1:100 dilution for cultured cells

  • Incubation: Overnight at 4°C for optimal results

  • Secondary antibody: Use appropriate fluorophore-conjugated secondary antibody at manufacturer's recommended dilution

• Co-localization Studies:

  • Consider co-staining with markers for:

    • Endosomal compartments (EEA1 for early endosomes, LAMP1 for late endosomes/lysosomes)

    • Vps33B to study interaction patterns

    • Other HOPS complex components

• Imaging Considerations:

  • VIPAS39 typically shows punctate cytoplasmic staining corresponding to endosomal structures

  • For detailed analysis of endosomal morphology, confocal or super-resolution microscopy is recommended

  • When studying mutations, compare wild-type and mutant patterns carefully for subtle changes in distribution

• Controls:

  • Negative control: Omit primary antibody

  • Positive control: Cell type with known VIPAS39 expression

  • Antibody validation: Consider siRNA knockdown of VIPAS39 to confirm specificity

VIPAS39 antibodies have been successfully used to demonstrate that pathogenic mutations can fragment VIPAS39-positive endosomes and alter subcellular localization of Vps33b to VIPAS39-positive endosomes .

How do mutations in VIPAS39 affect protein function and contribute to ARC syndrome pathogenesis?

Mutations in VIPAS39 contribute to ARC syndrome through multiple molecular mechanisms:

• Effects on VIPAS39-Vps33B interaction:

  • Some mutations, like nonsense mutations truncating VIPAS39 at position 220, abolish binding to Vps33B

  • Other mutations, such as truncation at position 425, retain Vps33B binding capability

  • This variability suggests multiple pathogenic mechanisms beyond simple disruption of protein-protein interaction

• Alterations in endosomal morphology and function:

  • Some VIPAS39 mutants cause fragmentation of VIPAS39-positive endosomes

  • All mutants alter the subcellular localization of Vps33b to VIPAS39-positive endosomes

  • These changes likely disrupt normal endosomal maturation or fusion processes

• Effects on polarized epithelial cells:

  • ARC syndrome is characterized by abnormalities in polarized liver and kidney cells

  • The VPS33B:VIPAS39 complex plays a role in the apical recycling pathway and maintenance of apical-basolateral polarity

  • Disruption of this function likely contributes to the liver and kidney manifestations of ARC syndrome

• Novel mutations and phenotypic variability:

  • Recently identified novel VIPAS39 pathological variants (c.762G > A; c.1064_1082delinsAGTG) are associated with variable clinical presentations

  • Patients with VIPAS39 mutations fall into severe and milder prognostic groups, with some reaching adolescence

  • This phenotypic variability suggests complex genotype-phenotype relationships

The pathogenesis of ARC syndrome appears to involve impaired VIPAS39 and Vps33b-dependent endosomal maturation or fusion, with VIPAS39 providing specificity to conserved endo-lysosomal tethers for fusion reactions among diverse endosomal compartments in metazoans .

What experimental approaches are most effective for studying VIPAS39's role in endosomal trafficking?

To effectively study VIPAS39's role in endosomal trafficking, researchers should consider the following experimental approaches:

• Protein-Protein Interaction Studies:

  • Yeast two-hybrid (Y2H) analysis: Effective for mapping interaction domains and testing effects of mutations

  • Co-immunoprecipitation: Can be enhanced with crosslinkers like DSP to stabilize interactions

  • Proximity labeling methods (BioID, APEX): To identify the broader VIPAS39 interactome

• Cellular Localization and Trafficking:

  • Immunofluorescence with co-localization analysis: To determine endosomal subtype localization

  • Live-cell imaging with fluorescently tagged proteins: For tracking dynamic processes

  • Electron microscopy with immunogold labeling: For ultrastructural analysis

• Functional Assays:

  • Cargo trafficking assays: Monitor transport of model cargo proteins through the endolysosomal system

  • Endosome fusion assays: To directly assess the role of VIPAS39 in membrane fusion events

  • pH-sensitive probes: To monitor endosomal maturation and acidification

• Genetic Manipulation:

  • CRISPR/Cas9 gene editing: Generate knockout cells or introduce specific mutations

  • Rescue experiments: Complement deficient cells with wild-type or mutant VIPAS39

  • Tissue-specific knockouts in model organisms: To study organ-specific phenotypes

• Disease-Relevant Models:

  • Patient-derived cells: Primary fibroblasts or iPSCs from ARC syndrome patients

  • Zebrafish models: Zebrafish vipas39 is expressed in liver and intestine, relevant to ARC syndrome pathology

  • Multi-cellular models: Organoids of liver or kidney to study effects on tissue organization

• Structural Biology:

  • Structural modeling of VIPAS39 and its complexes: Aids understanding of mutation effects

  • Analysis of conserved domains: The Vps16 C-terminal domain and spermatogenesis-defective protein 39 domain are key functional elements

These approaches should be combined to build a comprehensive understanding of VIPAS39 function, with careful selection of suitable controls and validation steps throughout the experimental process.

How can researchers design experiments to investigate the tissue-specific effects of VIPAS39 dysfunction?

The tissue-specific effects of VIPAS39 dysfunction, particularly in ARC syndrome, can be investigated through carefully designed experiments:

• Tissue-Specific Expression Analysis:

  • Gene expression databases and immunohistochemistry reveal VIPAS39 expression in multiple tissues, with notable expression in liver and intestine

  • Quantitative analysis of expression levels across tissues helps identify vulnerable cell populations

• Cell-Type-Specific Models:

  • For liver studies:

    • Primary hepatocytes or hepatic cell lines (HepG2, L02)

    • Liver organoids to recapitulate 3D tissue organization

    • Hepatocyte-specific conditional knockout animal models

  • For kidney studies:

    • Polarized renal epithelial cell models (MDCK cells)

    • Kidney-derived primary cells or cell lines

    • Podocyte-specific or tubular epithelium-specific gene manipulation

  • For neurological aspects:

    • Neuronal cultures or neuronal differentiation from iPSCs

    • Brain organoids to study neurodevelopmental effects

• Polarized Epithelial Cell Systems:

  • Culture cells on Transwell filters to establish apical-basolateral polarity

  • Measure transcytosis and protein sorting to different membrane domains

  • Analyze tight junction formation and epithelial barrier function

• Patient-Derived Materials:

  • Fibroblasts from ARC syndrome patients with VIPAS39 mutations

  • Generation of iPSCs and differentiation into relevant cell types

  • Comparison with cells from related healthy individuals as controls

• Comparative Studies Across Species:

  • C. elegans: SPE-39 function in multiple tissues including phagocytic coelomocytes and oocytes

  • Drosophila: carnation mutant phenotypes

  • Zebrafish: vipas39 expression in liver and intestine, with hepatobiliary system development effects

  • Mouse models: tissue-specific phenotypic analysis

• Systematic Phenotyping:

  • Comprehensive histological assessment of multiple tissues

  • Ultrastructural analysis of cellular organelles by electron microscopy

  • Functional assays specific to each tissue (e.g., bile acid transport for liver)

By integrating these approaches, researchers can develop a nuanced understanding of why certain tissues are particularly affected by VIPAS39 dysfunction, potentially revealing tissue-specific therapeutic targets for ARC syndrome.

What are common pitfalls in VIPAS39 antibody experiments and how can they be avoided?

When working with VIPAS39 antibodies, researchers should be aware of these common challenges and solutions:

Important considerations specific to VIPAS39:

• Molecular weight verification: VIPAS39 typically appears at 50 kDa on Western blots, despite a calculated molecular weight of 57 kDa

• Antibody validation: For definitive validation, use:

  • VIPAS39 knockout or knockdown samples as negative controls

  • Rescued knockout cells expressing wild-type VIPAS39 as positive controls

  • Multiple antibodies targeting different epitopes to confirm specificity

• Fixation sensitivity: Different fixation methods may affect epitope accessibility:

  • For immunofluorescence: 4% paraformaldehyde for 15-20 minutes typically works well

  • For tissue sections: Test both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

• Interaction studies: When studying VIPAS39-Vps33B interactions:

  • Consider using crosslinkers like DSP to stabilize potentially weak interactions

  • Include both wild-type and mutant controls when studying pathogenic mutations

  • Remember that not all pathogenic mutations disrupt the VIPAS39-Vps33B interaction

• Sample preparation: Handle samples carefully to preserve endosomal structure:

  • Avoid repeated freeze-thaw cycles

  • Process samples quickly to prevent protein degradation

  • Consider gentle fixation methods for preserving delicate membrane structures

By anticipating these challenges and implementing appropriate controls and optimizations, researchers can generate reliable and reproducible results in VIPAS39 experiments.

How can researchers optimize experimental design for studying VIPAS39 mutations?

When designing experiments to study VIPAS39 mutations, especially those associated with ARC syndrome, researchers should implement a comprehensive experimental design strategy:

• Selection of appropriate mutation models:

  • Patient-derived mutations (e.g., c.762G > A; c.1064_1082delinsAGTG)

  • Known functional mutations that affect VIPAS39-Vps33B binding (e.g., truncation at position 220)

  • Mutations that retain binding but affect other functions (e.g., truncation at position 425)

  • Novel mutations identified through genetic screening

• Expression system considerations:

  • Transient vs. stable expression: Stable expression allows long-term studies of cellular adaptation

  • Expression level control: Use inducible promoters to avoid artifacts from overexpression

  • Tagged vs. untagged constructs: Tags may interfere with function in some contexts

• Functional assays to assess mutation impact:

  • Protein-protein interactions: Y2H, co-IP, FRET/BRET

  • Subcellular localization: Immunofluorescence microscopy

  • Endosomal morphology: Assess fragmentation of VIPAS39-positive endosomes

  • Cargo trafficking: Monitor transport of model cargo through endolysosomal system

• Experimental controls:

  • Wild-type VIPAS39 as positive control

  • Empty vector as negative control

  • Multiple mutations representing different functional classes

  • Rescue experiments to confirm mutation-specific effects

• Advanced techniques for comprehensive analysis:

  • Structure-function analysis: Use molecular modeling to predict mutation effects

  • Interactome analysis: Determine how mutations affect the broader protein interaction network

  • Live-cell imaging: Monitor dynamic processes in real-time

  • Multi-parameter phenotypic analysis: Assess multiple cellular functions simultaneously

• Experimental design principles:

  • Use randomization and blinding where appropriate

  • Include sufficient biological and technical replicates

  • Control for extraneous variables that might influence results

  • Follow a systematic, hypothesis-driven approach

By implementing these experimental design strategies, researchers can generate robust data on how VIPAS39 mutations affect protein function and contribute to disease pathogenesis, potentially identifying therapeutic targets for ARC syndrome.

What emerging technologies might advance our understanding of VIPAS39 function?

Several cutting-edge technologies hold promise for deepening our understanding of VIPAS39 biology:

• Advanced Imaging Technologies:

  • Super-resolution microscopy: Techniques like STED, PALM, or STORM can resolve endosomal substructures beyond the diffraction limit

  • Correlative light and electron microscopy (CLEM): Combines the specificity of fluorescence with ultrastructural details

  • Live-cell lattice light-sheet microscopy: Enables long-term, high-resolution imaging of endosomal dynamics with minimal phototoxicity

• Proximity Labeling Technologies:

  • TurboID or miniTurbo: Faster biotin ligase variants for capturing transient interactions

  • Split-BioID: For studying compartment-specific interactions

  • APEX2-based proximity labeling: Alternative to biotin ligases with different spatiotemporal properties

• Structural Biology Advances:

  • Cryo-electron microscopy: To determine structures of VIPAS39-containing complexes

  • Integrative structural biology: Combining multiple techniques (X-ray, NMR, cross-linking) for complete structural models

  • AlphaFold2 and other AI-based structural prediction: For generating hypotheses about mutation effects

• Single-Cell Technologies:

  • Single-cell RNA-seq: To identify cell-type-specific effects of VIPAS39 dysfunction

  • Single-cell proteomics: To measure protein-level changes in rare cell populations

  • Spatial transcriptomics: To map VIPAS39-associated gene expression patterns in tissues

• Gene Editing and Screening:

  • CRISPR base editing: For precise introduction of patient mutations

  • CRISPR activation/inhibition: For tunable modification of VIPAS39 expression

  • CRISPR screens: To identify genetic modifiers of VIPAS39 function

• Organoid and Advanced Cell Culture:

  • Multi-organ-on-chip: To study inter-organ effects

  • Patient-derived organoids: For personalized disease modeling

  • Bioprinted tissues: For scalable 3D tissue models with controlled architecture

• Computational Approaches:

  • Systems biology: To model VIPAS39 in the context of entire trafficking networks

  • Machine learning: For image analysis and phenotype classification

  • Virtual screening: To identify small molecules that might stabilize mutant VIPAS39

These technologies, especially when applied in combination, have the potential to provide unprecedented insights into VIPAS39 function in normal biology and disease states.

How might research on VIPAS39 contribute to therapeutic approaches for ARC syndrome?

Research on VIPAS39 has significant potential to inform therapeutic strategies for ARC syndrome through several avenues:

• Genotype-Phenotype Correlations:

  • Recent studies have identified patients with milder ARC presentations, suggesting therapeutic potential for certain mutations

  • Understanding which mutations allow residual function could guide targeted therapies

  • Identification of naturally occurring compensatory mechanisms in less severely affected patients

• Small Molecule Approaches:

  • Protein stabilizers: For mutations that destabilize VIPAS39 but preserve function

  • Interaction enhancers: To strengthen weakened VIPAS39-Vps33B interactions

  • Chaperone molecules: To promote proper folding of mutant proteins

  • Read-through compounds: For nonsense mutations like p.Pro355_Thr361delinsGlnTer

• Gene Therapy Approaches:

  • VIPAS39 gene replacement: Particularly promising for liver and kidney manifestations

  • mRNA therapy: For transient expression in affected tissues

  • Antisense oligonucleotides: To modulate splicing for certain mutation types

• Cell-Based Therapies:

  • Hepatocyte transplantation: To address liver manifestations

  • iPSC-derived cell transplantation: Patient cells corrected ex vivo

  • Exosome-based approaches: To deliver functional VIPAS39 or correct downstream effects

• Pathway-Based Interventions:

  • Targeting compensatory trafficking pathways

  • Modulating downstream effects on apical-basolateral polarity

  • Addressing tissue-specific manifestations with organ-targeted approaches

• Early Intervention Strategies:

  • Improved genetic diagnosis enabling earlier intervention

  • Preventive measures for managing specific organ complications

  • Supportive therapies tailored to individual patient mutation profiles

The variability in clinical presentation of ARC syndrome patients with VIPAS39 mutations suggests potential therapeutic windows, particularly for those with milder phenotypes who survive beyond early childhood. Research focusing on understanding residual function in these cases could provide crucial insights for developing treatments that enhance this function or bypass the need for fully functional VIPAS39.