Recombinant Dog V-type proton ATPase subunit e 1 (ATP6V0E1)

What is ATP6V0E1 and what is its fundamental role in cellular function?

ATP6V0E1 is a component of vacuolar ATPase (V-ATPase), a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles. V-ATPase dependent organelle acidification is necessary for several critical intracellular processes, including protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation .

The V-ATPase complex consists of two major domains:

• The cytosolic V1 domain that hydrolyzes ATP

• The transmembrane V0 domain that translocates protons across membranes

ATP6V0E1 is considered part of the V0 domain, which consists of five different subunits (a, c, c', c", and d) . This protein plays an essential role in the structural integrity and functional efficiency of the V0 domain.

What is currently known about the genomic location and structure of dog ATP6V0E1?

The dog ATP6V0E1 gene has some interesting genomic features that researchers should consider:

• While radiation hybrid mapping initially localized the gene to chromosome 2 in dogs, genomic mapping indicates the functional gene is actually located in a region syntenic to human chromosome 5q35.3 .

• Notably, two non-transcribed pseudogenes related to ATP6V0E1 have been identified in dogs, which may complicate genetic analyses .

• The functional significance of these pseudogenes remains unclear, but their presence suggests evolutionary pressures that may be relevant to comparative studies across species.

How does ATP6V0E1 compare between dog and human orthologs?

When examining ATP6V0E1 across species, researchers should consider both conservation and divergence:

What experimental systems have been used to study ATP6V0E1 function?

Several model systems have been utilized to investigate ATP6V0E1 function:

• Zebrafish models: Zebrafish atp6v0e1-/- mutants have been developed and studied, particularly in relation to retinal function .

• Cell culture systems: Human cell lines (including HeLa cells with HIF-1α reporters) have been used to study ATP6V0E1 function .

• Canine models: Due to the presence of naturally occurring mutations in related V-ATPase genes in dogs, canine models have provided insights into V-ATPase complex function .

• Caenorhabditis elegans: This model organism has been used to study the effects of mutations in related V-ATPase genes .

Each system offers unique advantages for investigating different aspects of ATP6V0E1 biology, from cellular function to organism-level phenotypes.

What diseases or conditions are associated with ATP6V0E1 dysfunction?

While the search results don't specifically mention diseases directly linked to ATP6V0E1 mutations in dogs, related V-ATPase components have been associated with several conditions:

• Neurological disorders: Variants in ATP6V0A1 (another V-ATPase component) have been associated with progressive myoclonus epilepsy and developmental epileptic encephalopathy .

• Retinal dysfunction: Studies in zebrafish atp6v0e1-/- models have shown retinal abnormalities, suggesting a role in vision .

• V-ATPase-related disorders: The broader family of V-ATPase genes has been implicated in:

  • Osteopetrosis (ATP6V0A3)

  • Cutis laxa (ATP6V0A2)

  • Distal renal tubular acidosis (ATP6V1B1, ATP6V0A4)

  • Epileptic encephalopathy (ATP6V1A)

These associations indicate the importance of proper V-ATPase function in multiple organ systems and developmental processes.

How does ATP6V0E1 contribute to V-ATPase complex assembly and regulation?

ATP6V0E1 plays a crucial role in V-ATPase complex assembly and function through several mechanisms:

• Structural support: As a component of the V0 domain, ATP6V0E1 contributes to the structural integrity of the transmembrane portion of the complex .

• Regulated assembly: The V-ATPase complex efficacy depends on regulated assembly of the V1 sector onto the V0 sector. While the precise role of ATP6V0E1 in this process isn't fully characterized, it likely influences this dynamic association .

• Tissue-specific function: The use of specific V-ATPase subunits allows for specialized function in different tissues and cellular compartments. Some V-ATPase complexes localize to plasma membranes for extracellular acidification (in bone, kidney, and gut cells), while others remain intracellular for endosomal/lysosomal acidification .

Future research might explore how post-translational modifications of ATP6V0E1 affect these assembly processes and whether tissue-specific interacting partners influence its function in different cellular contexts.

What approaches can be used to study the impact of ATP6V0E1 mutations on cellular function?

Researchers investigating the functional consequences of ATP6V0E1 mutations can employ several complementary approaches:

• HIF-1α reporter systems: As demonstrated with other V-ATPase subunits, HIF-1α reporter cell lines can be used to assess how ATP6V0E1 mutations affect HIF-1α stability and activity. This provides insights into iron-dependent pathways regulated by V-ATPase .

• Organelle acidification assays: Since V-ATPase is critical for acidification of intracellular compartments, researchers can use pH-sensitive fluorescent probes to measure endosomal/lysosomal pH in cells with mutated ATP6V0E1 .

• Proteome profiling: Mass spectrometry-based proteomics can identify changes in protein expression resulting from ATP6V0E1 dysfunction. For example, in zebrafish atp6v0e1-/- models, proteome profiling revealed alterations in multiple pathways .

• Autophagy assessment: V-ATPase function is critical for autophagy. Markers such as LC3-II and p62 can be used to assess autophagic flux in cells with ATP6V0E1 mutations .

• Rescue experiments: Introducing wild-type ATP6V0E1 into mutant cells can confirm the specificity of observed phenotypes. This approach has been used successfully in similar V-ATPase studies .

How does ATP6V0E1 interact with histone deacetylase 6 (HDAC6) and what are the therapeutic implications?

Research in zebrafish has revealed an intriguing relationship between ATP6V0E1 and HDAC6:

• Therapeutic potential: Selective histone deacetylase 6 (HDAC6) inhibitors have been evaluated for their ability to preserve retinal morphology or restore vision in zebrafish atp6v0e1-/- models .

• Molecular mechanisms: Proteome profiling of atp6v0e1-/- zebrafish eyes treated with HDAC6 inhibitor Tubastatin A (TubA) revealed significant changes in protein expression patterns:

  • 23 proteins were upregulated and 50 proteins downregulated following treatment

  • Among the most downregulated proteins were retinoblastoma binding protein 9, tubulin alpha chain, and myosin regulatory light chain 2

• Pathway analysis: ClueGo and Cluepedia analyses of differentially expressed proteins in treated and untreated atp6v0e1-/- models identified multiple affected pathways, providing insights into disease mechanisms and therapeutic targets .

These findings suggest that HDAC6 inhibition may represent a promising therapeutic approach for conditions associated with ATP6V0E1 dysfunction, particularly those affecting retinal function.

What is known about the role of ATP6V0E1 in regulating autophagy and lysosomal function?

ATP6V0E1, as a component of the V-ATPase complex, plays a critical role in autophagy and lysosomal function through several mechanisms:

• Lysosomal acidification: The V-ATPase complex maintains the acidic pH of lysosomes, which is essential for the activity of lysosomal hydrolases. Dysfunction of ATP6V0E1 can impair this acidification, leading to failure of lysosomal hydrolysis .

• Autophagosome-lysosome fusion: Proper lysosomal acidification is required for efficient fusion of autophagosomes with lysosomes. ATP6V0E1 dysfunction may disrupt this process, resulting in autophagic dysfunction .

• CLEAR network regulation: ATP6V0A1, another V-ATPase component, is part of the Coordinated Lysosomal Expression and Regulation (CLEAR) network regulated by transcription factor EB (TFEB) . ATP6V0E1 may participate in similar regulatory networks, though this remains to be fully established.

• Disease relevance: The R740Q mutation in ATP6V0A1 leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction . Similar mechanisms may apply to ATP6V0E1 mutations.

This connection to autophagy and lysosomal function makes ATP6V0E1 a potential target for therapeutic interventions in neurodegenerative diseases and lysosomal storage disorders.

How does ATP6V0E1 expression change under different physiological and pathological conditions?

Although the search results don't provide comprehensive data on ATP6V0E1 expression changes across different conditions, several insights can be inferred:

• Tissue-specific expression: V-ATPase subunits often show tissue-specific expression patterns. For example, ATP6V0A1 is described as a "brain-enriched isoform" of the a subunit in the V0 domain . Similar tissue-specific expression patterns may exist for ATP6V0E1.

• Cancer relevance: Cancer cells can use specific V-ATPase subunits to activate oncogenic pathways. Research has investigated V-ATPase deregulation in aggressive gliomas, suggesting that ATP6V0E1 expression might be altered in certain cancers .

• Hypoxic conditions: Since V-ATPase inhibition affects HIF-1α levels, ATP6V0E1 may play a role in cellular responses to hypoxia . Changes in ATP6V0E1 expression under hypoxic conditions warrant further investigation.

• Developmental expression: Given the role of V-ATPase in fundamental cellular processes, ATP6V0E1 expression likely varies during development, particularly in tissues where it plays critical functions.

Future research using techniques like single-cell RNA sequencing could provide more detailed insights into ATP6V0E1 expression dynamics across different cell types, developmental stages, and disease states.

What are the optimal methods for expressing and purifying recombinant dog ATP6V0E1 protein?

Based on information from commercial providers and research practices, the following approach is recommended for expressing and purifying recombinant dog ATP6V0E1:

• Expression system selection:

  • Mammalian expression systems are preferred for ensuring proper folding and post-translational modifications

  • HEK293 or CHO cells are commonly used for expressing V-ATPase components

  • For structural studies requiring higher yields, insect cell systems (Sf9, Hi5) may be considered

• Vector design considerations:

  • State-of-the-art algorithms for plasmid design (Gene synthesis) are recommended

  • Addition of purification tags (His, FLAG, or GST) at either N- or C-terminus

  • Inclusion of protease cleavage sites for tag removal

  • Codon optimization for the expression host

• Purification strategy:

  • Initial capture using affinity chromatography (based on the chosen tag)

  • Intermediate purification using ion exchange chromatography

  • Final polishing using size exclusion chromatography

  • Buffer optimization to maintain protein stability (typically includes glycerol and reducing agents)

• Quality control:

  • SDS-PAGE to verify size and purity

  • Western blotting to confirm identity

  • Mass spectrometry to verify sequence and detect post-translational modifications

  • Functional assays to confirm biological activity

What experimental approaches can assess the functional integrity of recombinant ATP6V0E1?

To evaluate whether recombinant ATP6V0E1 maintains its native functionality, researchers can employ several complementary approaches:

• Integration into V-ATPase complexes:

  • Co-immunoprecipitation assays to verify interactions with other V-ATPase subunits

  • Blue native PAGE to assess incorporation into V-ATPase complexes

  • Reconstitution experiments in liposomes or nanodiscs

• Functional assays:

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • ATP hydrolysis assays when incorporated into full V-ATPase complexes

  • Binding assays with known interaction partners

• Structural validation:

  • Circular dichroism spectroscopy to assess secondary structure

  • Limited proteolysis to evaluate proper folding

  • Thermal shift assays to determine stability

• Cellular rescue experiments:

  • Introduction of recombinant ATP6V0E1 into ATP6V0E1-deficient cells

  • Assessment of V-ATPase-dependent processes (endosomal acidification, autophagy)

  • Rescue of phenotypes in animal models (such as zebrafish atp6v0e1-/- mutants)

These approaches collectively provide strong evidence for the functional integrity of recombinant ATP6V0E1 and its ability to participate in native V-ATPase complexes.

What tools and resources are available for detecting and analyzing ATP6V0E1 expression in tissue samples?

Researchers have several options for detecting and analyzing ATP6V0E1 expression in various experimental contexts:

• Antibody-based detection methods:

  • Immunohistochemistry protocols typically involve fixing samples in 4% PFA, quenching autofluorescence with 20 mM glycine, permeabilizing with 0.5% Triton, and blocking with 10% BSA

  • Primary antibody incubation is recommended overnight at 4°C with 1:100 dilution in PBS-BSA 10%

  • Secondary antibody incubation for 1 hour at room temperature with 1:1000 dilution

  • Commercial antibodies are available from several suppliers (Vector Laboratories, LSBio)

• Transcriptomic analysis:

  • RT-qPCR primers can be designed based on the dog ATP6V0E1 gene sequence

  • RNA-seq data analysis should account for potential pseudogene contamination

  • Single-cell RNA-seq can provide cell type-specific expression profiles

• Proteomic analysis:

  • Mass spectrometry-based proteomics has been successfully applied to detect ATP6V0E1 and related proteins in complex samples

  • Protein isolation from tissue samples can be performed using iST Sample Preparation Kit (PREOMICS)

  • LC-MS/MS analysis on systems like Thermo Scientific Q Exactive mass spectrometer connected to Dionex Ultimate 3000 RSLC nano

• Functional imaging:

  • V-ATPase activity can be visualized using pH-sensitive probes like LysoTracker

  • Live-cell imaging to monitor subcellular localization

What are the key considerations when designing CRISPR/Cas9-based knockout models for ATP6V0E1?

When creating CRISPR/Cas9-based knockout models for ATP6V0E1, researchers should consider several critical factors:

• Guide RNA design challenges:

  • The presence of pseudogenes in dogs complicates guide RNA design

  • Targeting unique regions that differ between the functional gene and pseudogenes is essential

  • In silico prediction of off-target effects should account for pseudogene sequences

  • Multiple guide RNAs targeting different exons may provide more reliable results

• Knockout strategy options:

  • Complete gene knockout may be lethal due to the essential nature of V-ATPase

  • Conditional knockout using Cre-lox or similar systems may be preferable

  • CRISPR interference (CRISPRi) for temporary knockdown might be valuable for initial studies

  • Consider introducing specific point mutations observed in human patients to model disease states

• Verification methods:

  • PCR-based genotyping should be designed to distinguish between the gene and pseudogenes

  • RT-qPCR to confirm reduction in mRNA levels

  • Western blotting to verify protein depletion

  • Sequencing to confirm the exact nature of the genetic modification

• Phenotypic assessment tools:

  • Lysosomal pH measurements to assess V-ATPase function

  • Autophagy assays (LC3, p62 staining)

  • Tissue-specific phenotypic assessments (e.g., retinal function tests based on zebrafish studies)

  • HIF-1α activity using reporter systems

How can proteomics be leveraged to understand ATP6V0E1 function in normal and disease states?

Proteomics offers powerful approaches to investigate ATP6V0E1 function and its dysregulation in disease:

• Interactome analysis:

  • Proximity labeling approaches (BioID, APEX) can identify ATP6V0E1 interaction partners

  • Co-immunoprecipitation followed by mass spectrometry can reveal stable interactors

  • Crosslinking mass spectrometry can capture transient interactions

• Global proteome changes:

  • Quantitative proteomics comparing wild-type and ATP6V0E1-deficient samples has been successful in zebrafish studies

  • iST Sample Preparation Kit (PREOMICS) has been used for protein isolation from eye tissues

  • Analysis on Thermo Scientific Q Exactive mass spectrometer connected to Dionex Ultimate 3000 chromatography system

  • Peptide separation on C18 home-made columns (C18RP Reposil-Pur, 100 × 0.075 mm × 3 μm)

• Pathway analysis:

  • ClueGo and Cluepedia plugins in Cytoscape have been used for pathway enrichment analysis

  • KEGG functional pathway databases provide valuable reference information

  • GO tree levels (min=3; max=8) and GO term restriction (min#genes=3, min%=1%) settings have been effective

  • Grouping using a Kappa Score Threshold of 0.4 has yielded meaningful results

• Post-translational modifications:

  • Phosphoproteomic analysis can reveal regulatory modifications on ATP6V0E1

  • Ubiquitylation studies may provide insights into protein turnover

  • Glycosylation analysis might reveal additional regulatory mechanisms

These proteomic approaches can identify molecular mechanisms underlying ATP6V0E1 function and potential therapeutic targets for associated diseases.

What therapeutic strategies targeting ATP6V0E1 or V-ATPase show promise for related disorders?

Several therapeutic approaches targeting V-ATPase components have shown promise in preclinical models:

• HDAC6 inhibition:

  • Selective HDAC6 inhibitors like Tubastatin A (TubA) have been evaluated for preserving retinal morphology or restoring vision in zebrafish atp6v0e1-/- models

  • This approach has shown promising results in restoring cone photoreceptor function

• Gene therapy approaches:

  • AAV-mediated gene delivery has been successful for other retinal disorders

  • Different promoters (mOP, hGRK1, CBA) have been evaluated for driving transgene expression in photoreceptors

  • Subretinal injection of AAV vectors has shown better results than intravitreal delivery for similar retinal conditions

• Small molecule modulators:

  • V-ATPase inhibitors like bafilomycin A1 are used as research tools but may have therapeutic potential

  • Iron supplementation may counteract some effects of V-ATPase dysfunction, as demonstrated in HIF-1α studies

• Targeting downstream pathways:

  • Modulating autophagy directly might bypass ATP6V0E1 dysfunction

  • HIF-1α pathway modulators could address secondary effects of V-ATPase impairment

These approaches provide a framework for developing therapies for conditions associated with ATP6V0E1 dysfunction, particularly retinal disorders and neurological conditions.

How might comparative studies of ATP6V0E1 across species inform therapeutic development?

Comparative analysis of ATP6V0E1 across species offers valuable insights for therapeutic development:

• Evolutionary conservation analysis:

  • Highly conserved regions likely represent functionally critical domains that should be preserved or targeted specifically

  • The first 24 amino acids, coded by exon 1, are highly conserved across 14 vertebrate species in similar V-ATPase genes, suggesting functional importance

• Natural variations as therapeutic clues:

  • Naturally occurring variations in dogs and other species may reveal compensatory mechanisms

  • Pseudogenes in dogs might contain valuable information about tolerated variations

• Cross-species disease modeling:

  • Zebrafish atp6v0e1-/- models have provided insights into retinal phenotypes and potential therapies

  • Caenorhabditis elegans models have demonstrated the impact of V-ATPase mutations on endolysosomal acidification

  • These complementary models can validate therapeutic targets across evolutionary distances

• Translation to human therapeutics:

  • Shared mutations between species provide confidence in therapeutic relevance

  • For example, the same homozygous mutation in PRCD (TGC → TAC) shows complete concordance with retinal disorders in 18 different dog breeds and was also identified in a human patient from Bangladesh

This comparative approach leverages natural genetic diversity to inform human therapeutic development while providing multiple model systems for preclinical testing.