Recombinant Bovine V-type proton ATPase subunit e 2 (ATP6V0E2)

What is the molecular function of ATP6V0E2 in bovine cells?

ATP6V0E2 functions as a subunit of the V-type proton ATPase complex, which is essential for acidification of intracellular compartments including lysosomes. This protein is part of the V0 domain, which forms the membrane-embedded proton channel. In bovine cells, ATP6V0E2 contributes to maintaining pH homeostasis across cellular compartments and plays a crucial role in lysosomal function.

Research indicates that ATP6V0E2 has a high knowledge value (0.91 on a 0-1 scale) regarding its molecular function . The protein is involved in hydrogen ion transmembrane transport and participates in the regulation of intracellular pH. Its activity is essential for various cellular processes including protein degradation, receptor-mediated endocytosis, and neurotransmitter release.

How does bovine ATP6V0E2 differ from human ATP6V0E2?

While both bovine and human ATP6V0E2 share significant sequence homology and functional similarities, several key differences exist:

Researchers should note that when studying bovine ATP6V0E2, species-specific tools and reagents may be required for optimal results. The functional consequences of these structural differences remain an active area of investigation.

What are the recommended methods for expressing recombinant bovine ATP6V0E2?

For successful expression of recombinant bovine ATP6V0E2, consider the following methodological approach:

• Expression System Selection: Mammalian expression systems (particularly HEK293 or CHO cells) are recommended due to their capacity for proper post-translational modifications. E. coli systems may be used for structural studies but may lack proper folding for functional analyses.

• Vector Design: Include a signal peptide appropriate for the expression system, along with a purification tag (His6 or FLAG) that won't interfere with the protein's function. Consider using the pET or pcDNA vector systems with appropriate promoters.

• Optimization Strategies:

  • Codon optimization for the expression host

  • Temperature reduction during induction (28-30°C)

  • Addition of chaperone proteins to improve folding

  • Use of fusion partners (e.g., MBP, SUMO) for enhanced solubility

• Purification Protocol: Implement a two-step purification process involving immobilized metal affinity chromatography followed by size exclusion chromatography to ensure high purity.

Note that ATP6V0E2 is a membrane-associated protein, which presents challenges for recombinant expression. Detergent screening (e.g., DDM, LMNG, or CHAPS) is often necessary to identify optimal conditions for solubilization and purification.

What experimental controls should be included when studying ATP6V0E2 function?

When designing experiments to study ATP6V0E2 function, include the following controls:

• Positive Controls:

  • Well-characterized V-ATPase inhibitors (e.g., bafilomycin A1 or concanamycin A)

  • Known lysosomal function modulators (e.g., chloroquine for comparison)

  • Related V-ATPase subunits with established functions

• Negative Controls:

  • Empty vector transfections

  • Non-targeting siRNA/shRNA for knockdown studies

  • Inactive mutants of ATP6V0E2 (e.g., site-directed mutants at critical residues)

• Expression Verification:

  • Western blot validation of protein expression

  • qRT-PCR for transcript level confirmation

  • Immunofluorescence for localization confirmation

• Functional Validation:

  • Lysosomal pH measurements (using LysoSensor probes)

  • ATPase activity assays

  • Protein-protein interaction controls (pull-down with other V-ATPase components)

Recent studies suggest that anlotinib treatment upregulates ATP6V0E2 and other lysosome-related genes . When studying drug effects on ATP6V0E2, including appropriate vehicle controls and time-course analyses is essential.

How does ATP6V0E2 contribute to lysosomal function in bovine cells and how can this be experimentally assessed?

Experimental assessment methodology:

• Lysosomal Acidification Assays:

  • Use pH-sensitive fluorescent probes (LysoSensor Yellow/Blue DND-160) to measure lysosomal pH

  • Implement live-cell imaging with ratiometric analysis

  • Compare wild-type cells with ATP6V0E2-knockdown/knockout cells

• Enzyme Activity Measurements:

  • Assess activities of lysosomal enzymes (e.g., cathepsins, β-hexosaminidase) using fluorogenic substrates

  • Compare enzyme activities across pH gradients to determine pH optima shifts

• Autophagy Flux Analysis:

  • Monitor LC3-II/I ratio by western blotting

  • Assess autophagic flux using tandem-fluorescent LC3 (tf-LC3) reporters

  • Quantify autophagosome-lysosome fusion events using co-localization studies

Research has shown that anlotinib activates lysosomal function and enhances the fusion of autophagosomes and lysosomes . When ATP6V0E2 is knocked down, this enhanced lysosomal function is attenuated, suggesting its critical role in this process. Similar experimental approaches can be applied to bovine systems to elucidate species-specific functions.

What is the relationship between ATP6V0E2 and the mTOR signaling pathway in bovine cells?

The relationship between ATP6V0E2 and mTOR signaling represents an important regulatory axis in cellular homeostasis. Current research indicates that:

• Lysosomal Positioning and mTOR Recruitment:
  ATP6V0E2, as part of the V-ATPase complex, helps establish the acidic lysosomal environment where mTOR signaling is regulated. Evidence suggests that V-ATPase interacts with the Ragulator complex, which is essential for mTOR recruitment to lysosomes.

• Bidirectional Regulation:

  • mTOR inhibition can influence ATP6V0E2 expression and V-ATPase assembly

  • ATP6V0E2 function affects mTOR activation status

• TFEB Regulation:
  Anlotinib treatment inhibits mTOR signaling and activates TFEB (Transcription Factor EB), a key nuclear transcription factor controlling lysosome biogenesis and function . This activation promotes TFEB nuclear translocation and enhances its transcriptional activity, including potentially regulating ATP6V0E2.

Experimental approaches to study this relationship:

When studying bovine systems, it's important to validate all antibodies and reagents for cross-reactivity with bovine proteins, as many commercial tools are optimized for human or mouse samples.

How can gene editing approaches be optimized for studying ATP6V0E2 function in bovine cells?

Gene editing approaches offer powerful tools for studying ATP6V0E2 function, but require specific optimization for bovine systems:

• CRISPR-Cas9 System Optimization:

  • Design bovine-specific gRNAs targeting ATP6V0E2 using bovine genome references

  • Screen multiple gRNAs for efficiency (3-5 targets per region of interest)

  • Optimize Cas9 expression in bovine cells using species-appropriate promoters

  • Consider using Cas9 nickase for reduced off-target effects

• Delivery Methods for Bovine Cells:

  • Nucleofection typically yields higher efficiency than lipofection for primary bovine cells

  • Lentiviral delivery may be preferred for difficult-to-transfect cells

  • For primary bovine cells, electroporation parameters must be specifically optimized (typical settings: 250V, 950μF for fibroblasts)

• Verification Strategies:

  • T7 endonuclease assay for initial editing efficiency

  • Sanger sequencing of PCR products spanning the target site

  • Western blot verification of protein knockout/knockdown

  • Functional validation using lysosomal pH measurements

• Rescue Experiments:

  • Re-express wild-type or mutant bovine ATP6V0E2 in knockout cells

  • Use species-matched expression constructs

  • Consider inducible expression systems to control timing of rescue

For studying bovine ATP6V0E2, creating cell lines with conditional knockout systems may be preferable, as complete loss of V-ATPase function can be lethal to cells. Inducible CRISPR systems or degron-based approaches offer temporal control over protein depletion.

What are the experimental approaches to investigate the role of ATP6V0E2 in autophagy and its potential therapeutic implications?

ATP6V0E2, as a component of the V-ATPase complex, plays a crucial role in autophagy through its involvement in lysosomal acidification. Research investigating its role in autophagy should consider the following approaches:

• Autophagy Flux Measurement:

  • Monitor LC3-II accumulation with and without lysosomal inhibitors

  • Use tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to visualize autophagosome-lysosome fusion

  • Quantify p62/SQSTM1 levels as autophagy substrate

• ATP6V0E2 Modulation Strategies:

  • Genetic: CRISPR knockout, siRNA knockdown, overexpression

  • Pharmacological: V-ATPase inhibitors (bafilomycin A1, concanamycin A)

  • Compare effects of global V-ATPase inhibition versus specific ATP6V0E2 targeting

• Functional Readouts:

  • Measurement of autophagic degradation of long-lived proteins

  • Assessment of mitophagy using MitoTracker and LC3 co-localization

  • Electron microscopy to visualize autophagic structures

• Therapeutic Assessment Models:

  • Stress response evaluation (nutrient deprivation, oxidative stress)

  • Drug combination studies (with autophagy modulators)

  • Cell viability and apoptosis measurements

Research has shown that anlotinib treatment activates lysosomal function via inhibiting mTOR signaling and enhancing TFEB transcriptional activity . When TFEB or ATP6V0E2 are knocked down, the enhanced lysosomal function and autophagy induced by anlotinib are attenuated. Furthermore, inhibition of lysosomal function enhances anlotinib-induced cell death and tumor suppression, which may be attributed to high levels of reactive oxygen species (ROS) .

This suggests a potential therapeutic strategy where modulation of ATP6V0E2 activity could be combined with other treatments to enhance anticancer effects. Similar approaches could be explored in bovine systems to understand species-specific responses.

How do post-translational modifications affect bovine ATP6V0E2 function and stability?

Post-translational modifications (PTMs) significantly impact ATP6V0E2 function and stability. Understanding these modifications is crucial for comprehensive characterization of this protein:

• Common PTMs Affecting ATP6V0E2:

  • Phosphorylation: May regulate assembly/disassembly of V-ATPase complex

  • Glycosylation: Potentially affects protein stability and trafficking

  • Ubiquitination: Regulates protein turnover and degradation

• Experimental Approaches to Study PTMs:

• PTM Dynamics Assessment:

  • Pulse-chase experiments to measure protein half-life

  • Cycloheximide chase assays for degradation kinetics

  • Stimulus-dependent changes in modification status

• Comparative Analysis:

  • Compare bovine ATP6V0E2 PTM profile with human and other species

  • Identify conserved vs. species-specific modification sites

  • Correlate differences in PTMs with functional variations

The high knowledge value score (0.91) for ATP6V0E2 molecular function suggests detailed understanding of its basic mechanisms, but species-specific PTM patterns in bovine ATP6V0E2 represent an important area for further investigation. Researchers should employ a combination of biochemical and proteomic approaches to comprehensively characterize these modifications.

What are common challenges in generating high-yield recombinant bovine ATP6V0E2 and how can they be addressed?

Producing high-yield recombinant bovine ATP6V0E2 presents several challenges due to its membrane-associated nature. Here are common issues and solutions:

• Protein Misfolding and Aggregation:

  • Challenge: ATP6V0E2 may form inclusion bodies in bacterial systems

  • Solution: Express at lower temperatures (16-18°C), use solubility-enhancing tags (SUMO, MBP), or switch to eukaryotic expression systems

• Low Expression Levels:

  • Challenge: Membrane proteins often express poorly

  • Solution: Optimize codon usage for expression host, use strong inducible promoters, screen multiple expression constructs with varied N/C-terminal tags

• Protein Instability:

  • Challenge: Rapid degradation after expression

  • Solution: Add protease inhibitors throughout purification, identify and modify unstable regions, optimize buffer conditions

• Proper Detergent Selection:

  • Challenge: Finding detergents that maintain protein solubility without denaturing

  • Solution: Systematic screening of detergents (start with mild detergents like DDM, LMNG, and digitonin)

Optimization Strategy Table:

For functional studies, consider reconstituting purified protein into liposomes or nanodiscs to maintain native-like membrane environment.

What are the best approaches for detecting ATP6V0E2 in bovine tissue samples?

Detecting ATP6V0E2 in bovine tissues requires careful consideration of detection methods and sample preparation:

• Antibody-Based Detection:

  • Western Blotting: Use antibodies verified for bovine specificity; optimize extraction buffers for membrane proteins (containing 1% digitonin or 0.5% DDM)

  • Immunohistochemistry (IHC): Test multiple antigen retrieval methods; citrate buffer (pH 6.0) often works well for membrane proteins

  • Immunofluorescence: Use Triton X-100 permeabilization; consider tyramide signal amplification for low abundance detection

• Transcript Detection:

  • qRT-PCR: Design bovine-specific primers spanning exon junctions

  • RNA-Seq: Use specific alignment to bovine genome (not human reference)

  • In situ hybridization: Design species-specific probes targeting unique regions

• Sample Preparation Considerations:

  • Fresh tissue provides better results than frozen for protein detection

  • For membrane proteins, avoid harsh detergents that may destroy epitopes

  • Optimize fixation time for IHC (overfixation can mask epitopes)

• Control Recommendations:

  • Include tissues with known high and low expression

  • Use siRNA knockdown samples as negative controls

  • Consider recombinant protein as positive control

When working with bovine tissues, validation of reagents is critical as many commercial antibodies are developed against human proteins. Cross-reactivity should be experimentally confirmed before proceeding with extensive studies.

How can researchers troubleshoot inconsistent results in ATP6V0E2 functional assays?

Functional assays for ATP6V0E2 may yield inconsistent results due to various factors. Here's a systematic troubleshooting approach:

• Common Sources of Variability:

  • Cell confluence effects on V-ATPase expression

  • Passage number influencing cellular phenotypes

  • Buffer composition affecting enzyme activity

  • Incomplete protein complex assembly

• Assay-Specific Troubleshooting:

• Validation Strategies:

  • Use multiple methodological approaches to measure the same parameter

  • Include positive controls (V-ATPase inhibitors) in each experiment

  • Establish dose-response relationships to confirm specificity

  • Validate key findings with both gain- and loss-of-function approaches

• Technical Considerations:

  • Standardize cell culture conditions (media lot, serum source)

  • Establish consistent protein extraction protocols

  • Document reagent sources and lot numbers

  • Implement blinded analysis where possible

Research has shown that knocking down ATP6V0E2 attenuates enhanced lysosomal function induced by treatments like anlotinib . If similar inconsistencies are observed in bovine systems, consider testing the efficiency of your knockdown/knockout system and validating the specificity of your targeting approach.

What are the emerging roles of ATP6V0E2 in cellular signaling beyond lysosomal acidification?

Recent research has revealed that ATP6V0E2 and other V-ATPase components have functions beyond their canonical role in lysosomal acidification:

• Signaling Platform Functions:

  • Evidence suggests V-ATPase components may act as scaffolds for signaling complexes

  • ATP6V0E2 may participate in protein-protein interactions independent of its role in the V-ATPase complex

  • Potential involvement in non-canonical signaling pathways

• Metabolic Regulation:

  • Emerging evidence links V-ATPase activity to cellular metabolism

  • ATP6V0E2 may influence AMPK signaling through lysosomal positioning

  • Potential role in nutrient sensing mechanisms

• Transcriptional Regulation:

  • Research suggests that ATP6V0E2 expression is regulated by TFEB, a master regulator of lysosomal biogenesis

  • Potential feedback loops between V-ATPase activity and transcriptional programs

• Cell Death Pathways:

  • V-ATPase inhibition enhances cell death in combination with other treatments

  • ATP6V0E2 may influence the balance between protective autophagy and cell death

  • Modulation of ATP6V0E2 affects reactive oxygen species levels, potentially influencing cell fate decisions

Future research should employ proximity labeling techniques (BioID, APEX) to identify novel ATP6V0E2 interacting partners that may reveal additional signaling functions beyond its role in the V-ATPase complex.

How does ATP6V0E2 contribute to species-specific differences in cellular physiology between bovine and human systems?

Understanding species-specific differences in ATP6V0E2 function provides valuable insights for comparative physiology and translational research:

• Evolutionary Conservation Analysis:

  • Sequence alignment shows conserved functional domains across species

  • Species-specific variations occur primarily in regulatory regions

  • Potential differences in post-translational modification sites

• Expression Pattern Differences:

  • Bovine tissues may show different ATP6V0E2 expression patterns compared to human counterparts

  • Tissue-specific isoform usage may vary between species

  • Developmental regulation may follow species-specific patterns

• Functional Implications:

  • Differences in pH regulation may reflect metabolic and physiological adaptations

  • Species-specific interactions with regulatory proteins

  • Potential variations in drug sensitivity and inhibitor binding

Research Methodologies for Cross-Species Comparison:

While the knowledge value regarding molecular function of ATP6V0E2 is high (0.91) , species-specific variations represent an important area for further investigation. These differences may have significant implications for using bovine models in research relevant to human health.

What is the current understanding of ATP6V0E2 in the context of disease models and potential therapeutic applications?

Recent research has begun to uncover the significance of ATP6V0E2 in various disease contexts:

• Cancer Biology:

  • Transcriptome sequencing analysis reveals that anlotinib treatment upregulates ATP6V0E2 and other lysosome-related genes in human colon cancer

  • ATP6V0E2 appears involved in regulating lysosomal function, which affects cancer cell survival

  • Knockdown of ATP6V0E2 attenuates enhanced lysosomal function and autophagy induced by anlotinib treatment

  • Inhibition of lysosomal function enhances anlotinib-induced cell death and tumor suppression, potentially through increased reactive oxygen species levels

• Potential Therapeutic Approaches:

  • Targeting ATP6V0E2 may enhance the efficacy of certain anti-cancer treatments

  • Combined treatment with V-ATPase inhibitors and other therapeutic agents shows synergistic effects

  • Modulation of ATP6V0E2 activity could affect autophagy-dependent resistance mechanisms

• Biomarker Potential:

  • Expression levels of ATP6V0E2 may serve as prognostic indicators

  • Changes in ATP6V0E2 activity might predict response to specific therapies

  • ATP6V0E2-AS1, a long non-coding RNA, has been implicated in protein translation regulation in prostate cancer

• Methodological Considerations for Disease Models:

  • Cell line selection should reflect the tissue of interest

  • Patient-derived xenografts may better capture disease heterogeneity

  • Consideration of microenvironmental factors affecting V-ATPase function

While much of this research has focused on human disease models, similar approaches could be applied to investigate bovine ATP6V0E2 in the context of bovine diseases or as a comparative model system.

What advanced imaging techniques are most effective for studying ATP6V0E2 localization and dynamics in bovine cells?

Studying the localization and dynamics of ATP6V0E2 requires sophisticated imaging approaches tailored to membrane proteins:

• Super-Resolution Microscopy Techniques:

  • Stimulated Emission Depletion (STED) microscopy: Achieves resolution of ~30-50 nm

  • Photoactivated Localization Microscopy (PALM): Ideal for tagged proteins, resolution ~10-20 nm

  • Stochastic Optical Reconstruction Microscopy (STORM): Excellent for immunolabeled endogenous proteins

• Live-Cell Imaging Approaches:

  • Fluorescent Protein Tagging: Consider monomeric variants (mEGFP, mCherry) with small linkers

  • Optimal Tag Placement: N-terminal tagging generally preferred for ATP6V0E2

  • Photoactivatable/Photoconvertible Proteins: For pulse-chase visualization of protein pools

• Colocalization Analysis:

  • Multi-channel Imaging: Combine ATP6V0E2 labeling with organelle markers

  • Quantitative Colocalization: Use Pearson's or Mander's coefficients

  • Super-Resolution Colocalization: Consider proximity ligation assays for protein-protein interactions

• Dynamic Studies:

  • Fluorescence Recovery After Photobleaching (FRAP): Assess protein mobility

  • Fluorescence Loss In Photobleaching (FLIP): Examine continuity of protein pools

  • Single Particle Tracking: Monitor individual complex movement

Technical Considerations for Bovine Cells:

When studying ATP6V0E2 localization, it's crucial to distinguish between the protein's localization when assembled in the V-ATPase complex versus when it exists as a free subunit, as these pools may have distinct functional significance.

What are the key knowledge gaps in ATP6V0E2 research and recommended approaches to address them?

Despite advances in understanding ATP6V0E2, several significant knowledge gaps remain:

• Structural Understanding:

  • Gap: Limited structural information on bovine ATP6V0E2, particularly in the context of the assembled V-ATPase complex

  • Recommendation: Apply cryo-electron microscopy to determine high-resolution structures of bovine V-ATPase with focus on the ATP6V0E2 subunit

• Regulatory Mechanisms:

  • Gap: Incomplete understanding of how ATP6V0E2 expression and function are regulated in bovine tissues

  • Recommendation: Perform comprehensive promoter analysis and investigate transcription factor binding sites specific to bovine ATP6V0E2

• Tissue-Specific Functions:

  • Gap: Limited data on tissue-specific roles of ATP6V0E2 in bovine physiology

  • Recommendation: Develop tissue-specific conditional knockout models to evaluate physiological impact

• Interactome Characterization:

  • Gap: Incomplete mapping of ATP6V0E2 protein-protein interactions in bovine cells

  • Recommendation: Implement BioID or APEX2 proximity labeling to identify tissue-specific and condition-specific interacting partners

• Comparative Biology:

  • Gap: Limited comparative analysis between bovine and human ATP6V0E2 function

  • Recommendation: Perform systematic cross-species complementation studies to identify functionally conserved and divergent properties

Research has shown that ATP6V0E2 is involved in lysosomal function and may be regulated through the mTOR-TFEB axis . Further investigation into the regulatory mechanisms controlling ATP6V0E2 expression and activity in bovine systems will be particularly valuable.

What collaborative research approaches would accelerate progress in understanding bovine ATP6V0E2 function?

Advancing research on bovine ATP6V0E2 would benefit significantly from multidisciplinary collaborative approaches:

• Integrative Omics Approaches:

  • Combination of proteomics, transcriptomics, and metabolomics to understand ATP6V0E2 in system-wide context

  • Integration of data across multiple bovine tissues and developmental stages

  • Computational modeling of regulatory networks involving ATP6V0E2

• Cross-Disciplinary Collaboration Opportunities:

• Technology Development Needs:

  • Bovine-specific CRISPR libraries for high-throughput screening

  • Improved antibodies and nanobodies specific for bovine ATP6V0E2

  • Organoid systems representing bovine tissues for functional studies

• Data Sharing and Infrastructure:

  • Establishment of bovine tissue-specific expression atlases

  • Repositories for bovine-specific reagents and protocols

  • Standardized reporting of experimental conditions for bovine cell culture

As research moves forward, combining expertise across molecular biology, structural biology, cell physiology, and veterinary medicine will be essential to fully elucidate the functions of bovine ATP6V0E2 and translate this knowledge into practical applications.