Recombinant Mouse V-type proton ATPase subunit e 2 (Atp6v0e2)

What is Atp6v0e2 and what is its function in cellular systems?

Atp6v0e2 (ATP6V0E2 in humans) is a protein-coding gene that encodes the e2 subunit of the V0 domain in vacuolar-type H+-ATPase (V-ATPase). V-ATPase functions as a multisubunit enzyme transporter that acidifies intracellular compartments in eukaryotic cells. The complex consists of two major functional domains: the V1 domain, which hydrolyzes ATP, and the V0 domain, which translocates protons across membranes. Specifically, the e2 subunit is a small, hydrophobic component of the V0 domain that interacts with other subunits including a1 and subunit f, providing structural support to the complex .

V-ATPase is ubiquitously expressed and found in various organelles including vacuoles, lysosomes, endosomes, clathrin-coated vesicles, and synaptic vesicles. In specialized cells, V-ATPase can also be found in plasma membranes where it contributes to processes like urinary acidification, bone resorption, and sperm maturation .

How does Atp6v0e2 differ from other V-ATPase subunits?

Atp6v0e2 is distinguished from other V-ATPase subunits in several ways:

Unlike catalytic subunits that directly participate in ATP hydrolysis or proton translocation, the e2 subunit appears to play a primarily structural role. Recent high-resolution cryo-EM studies have revealed that subunit e2 interacts with specific residues including Arg77, Phe78, Leu79, and Glu81, which form important connections with subunit a1 and subunit f .

What methods are most effective for purifying recombinant mouse Atp6v0e2 protein?

Purification of recombinant mouse Atp6v0e2 presents significant challenges due to its small size and hydrophobic nature. The most effective approach combines bacterial expression systems with specialized solubilization and purification techniques:

• Expression system selection: E. coli BL21(DE3) with a pET expression system incorporating a fusion tag (His6, GST, or MBP) to enhance solubility and facilitate purification.

• Induction conditions: Low-temperature induction (16-18°C) with reduced IPTG concentration (0.1-0.5 mM) over extended periods (16-20 hours) to minimize inclusion body formation.

• Membrane protein solubilization:

  • Primary solubilization with detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin

  • Alternative approach using styrene-maleic acid (SMA) copolymers that extract proteins with their native lipid environment

• Purification protocol:

  • Initial IMAC (immobilized metal affinity chromatography) for His-tagged proteins

  • Size exclusion chromatography to remove aggregates and purify properly folded protein

  • Optional tag removal using specific proteases, followed by a second IMAC step

For functional studies, reconstitution into liposomes or nanodiscs may be necessary to maintain the native conformation and activity of this membrane protein .

What are the optimal conditions for detecting Atp6v0e2 in native tissue samples?

Detecting Atp6v0e2 in native tissue samples presents challenges due to its small size and hydrophobic nature. Recent high-resolution cryo-EM studies of V-ATPase in synaptic vesicles noted that the small and hydrophobic subunits e2 and f were not detected, highlighting this difficulty . Researchers should employ a combination of techniques to optimize detection:

• Sample preparation:

  • Fresh tissue preservation with minimal processing time

  • Gentle membrane solubilization using mild detergents like digitonin (0.5-1%)

  • Avoiding harsh reducing conditions that may disrupt structural integrity

• Western blotting optimization:

  • Transferring proteins to PVDF membranes (rather than nitrocellulose) for better retention of hydrophobic proteins

  • Extended transfer times (overnight at low voltage)

  • Using specialized antibodies with verified specificity for the e2 subunit

  • Enhanced chemiluminescence detection with prolonged exposure times

• Mass spectrometry approaches:

  • Enrichment of membrane fractions before analysis

  • Cross-linking mass spectrometry (XL-MS) to capture interactions with partner proteins

  • Special consideration for hydrophobic peptides in data analysis

• Immunohistochemistry considerations:

  • Antigen retrieval optimization specific for membrane proteins

  • Using a combination of antibodies against Atp6v0e2 and other V-ATPase subunits

  • Super-resolution microscopy techniques for improved visualization

When possible, validation using tissues from Atp6v0e2 knockout models as negative controls significantly enhances confidence in detection results .

How can researchers effectively express and characterize functional Atp6v0e2 in vitro?

To express and characterize functional Atp6v0e2 in vitro, researchers should adopt a systematic approach:

• Expression system selection:

  • Mammalian cell lines (HEK293, CHO) for proper post-translational modifications

  • Insect cell systems (Sf9, High Five) for higher yields of functional membrane proteins

  • Cell-free expression systems when rapid screening is needed

• Vector design considerations:

  • Incorporate fluorescent tags (GFP, mCherry) for localization studies

  • Include epitope tags (FLAG, HA) positioned to minimize functional interference

  • Consider inducible expression systems to control expression levels

• Functional characterization protocol:

  • Proton translocation assays using pH-sensitive fluorescent dyes (BCECF, pHrodo)

  • Bafilomycin A1 sensitivity assays to confirm V-ATPase-specific activity

  • Co-immunoprecipitation studies to verify incorporation into the V-ATPase complex

  • ATPase activity measurements in purified or enriched preparations

• Reconstitution approaches:

  • Proteoliposome preparation with defined lipid composition

  • Nanodiscs for single-molecule studies

  • Co-expression with other V0 domain subunits for proper complex assembly

For characterizing the structural role of Atp6v0e2, researchers can employ site-directed mutagenesis targeting the interaction residues (e.g., Arg77, Phe78, Leu79, Glu81) identified in recent structural studies, followed by assembly assays to determine the impact on V-ATPase complex integrity .

What are the recommended controls and validation steps for Atp6v0e2 antibody specificity?

Ensuring antibody specificity is critical for Atp6v0e2 research. A comprehensive validation approach should include:

Additionally, researchers should document and report the catalog number, lot number, dilution used, and specific experimental conditions to enhance reproducibility. When using commercial antibodies, validation data provided by manufacturers should be critically evaluated and supplemented with laboratory-specific validation when possible .

How does Atp6v0e2 contribute to V-ATPase assembly and regulation in different cellular contexts?

Atp6v0e2 plays subtle but important roles in V-ATPase assembly and regulation that can vary across cellular contexts:

• Structural contributions:

  • Recent structural studies reveal that Atp6v0e2 forms specific interactions with subunit a1 and subunit f, creating a network of interactions that stabilize the V0 domain architecture .

  • The protein's interactions with key residues (Arg482 and Glu492 from subunit a1; Pro55 and Tyr59 from subunit f) suggest it helps maintain proper alignment of the proton-conducting elements .

• Tissue-specific regulation:

  • While Atp6v0e1 is ubiquitously expressed, Atp6v0e2 shows more tissue-restricted expression patterns, suggesting specialized functions in certain cell types.

  • In specialized epithelial cells, Atp6v0e2 may contribute to apical-basal polarity establishment and maintenance, as V-ATPase function has been shown crucial for epithelial organization during embryogenesis .

• Assembly dynamics:

  • Atp6v0e2 is likely incorporated during the early stages of V0 domain assembly in the endoplasmic reticulum.

  • The assembly process involves step-wise incorporation of subunits, with Atp6v0e2 potentially playing a role in facilitating proper insertion of larger subunits like subunit a1.

• Isoform-specific regulation:

  • The existence of multiple e subunit isoforms (e1 and e2) suggests differential regulation and potentially distinct functional properties in different cellular contexts.

  • These isoforms may influence V-ATPase assembly efficiency, subcellular localization, or interaction with regulatory proteins in a cell-type-specific manner.

Research using targeted mutations in the interacting residues of Atp6v0e2 could further elucidate its precise role in V-ATPase assembly and function within specific cellular contexts .

What is the relationship between Atp6v0e2 expression and lysosomal function in pathological conditions?

Recent research has revealed intriguing connections between Atp6v0e2 expression and lysosomal function in disease states:

• Cancer biology:

  • Transcriptome sequencing analysis in human colon cancer cells treated with the tyrosine kinase inhibitor anlotinib showed significant upregulation of ATP6V0E2 expression .

  • This upregulation coincided with enhanced lysosomal function and autophagy, suggesting ATP6V0E2 may be a key mediator of lysosomal activation in response to treatment .

  • Importantly, knockdown of ATP6V0E2 attenuated anlotinib-induced lysosomal activation, confirming its functional significance in this pathway .

• Mechanistic pathways:

  • Anlotinib treatment inhibits mTOR signaling and activates TFEB (Transcription Factor EB), a master regulator of lysosomal biogenesis.

  • TFEB activation promotes its nuclear translocation and enhances transcriptional activity of lysosomal genes, including ATP6V0E2 .

  • This creates a regulatory circuit where mTOR inhibition → TFEB activation → ATP6V0E2 upregulation → enhanced lysosomal function.

• Therapeutic implications:

  • Paradoxically, inhibition of lysosomal function enhanced anlotinib-induced cell death and tumor suppression, potentially due to increased reactive oxygen species (ROS) levels .

  • This suggests a complex role where ATP6V0E2-mediated lysosomal activation may initially serve as a cellular survival mechanism against therapeutic stress.

These findings suggest that ATP6V0E2 expression levels could serve as a potential biomarker for treatment response and that combinatorial approaches targeting both cancer cells and lysosomal function might enhance therapeutic efficacy .

How does Atp6v0e2 function differ from its paralog Atp6v0e1 in experimental models?

While both Atp6v0e1 and Atp6v0e2 are e subunits of the V0 domain, they exhibit important differences in expression patterns, functional properties, and experimental characteristics:

• Expression patterns:

  • Atp6v0e1 is ubiquitously expressed across tissues and considered the "housekeeping" e subunit.

  • Atp6v0e2 shows a more restricted expression pattern and may be upregulated under specific conditions, such as during cancer treatment with anlotinib .

• Genetic redundancy:

  • Studies suggest incomplete redundancy between these paralogs.

  • While Atp6v0e1 knockout causes more severe phenotypes, Atp6v0e2 knockout may produce subtler, tissue-specific effects that become apparent under stress conditions.

• Structural contributions:

  • High-resolution structural studies have begun to reveal specific interactions formed by Atp6v0e2 with other V-ATPase subunits, including important connections with subunit a1 and subunit f .

  • These interactions involve specific residues (Arg77, Phe78, Leu79, and Glu81) that may differ from the corresponding interactions formed by Atp6v0e1 .

• Functional specialization:

  • Atp6v0e2 may have evolved specialized functions in certain cell types or under specific conditions.

  • For example, in cancer cells, ATP6V0E2 upregulation appears to be particularly important for lysosomal function enhancement in response to anticancer treatments .

• Experimental detection:

  • Both paralogs present detection challenges due to their small size and hydrophobicity.

  • Development of paralog-specific antibodies requires careful design and validation to avoid cross-reactivity.

Researchers investigating these paralogs should consider employing paralog-specific knockout models, rescue experiments with the alternate paralog, and careful analysis of expression patterns across different tissues and experimental conditions to fully understand their distinct and overlapping functions .

What are common challenges in detecting Atp6v0e2 in experimental systems and how can they be addressed?

Detection of Atp6v0e2 presents several technical challenges that researchers frequently encounter:

Researchers should document and report detailed methodological approaches, including specific buffer compositions, antibody validation data, and controls used, to enhance reproducibility across studies .

How can researchers effectively use recombinant Atp6v0e2 in protein interaction studies?

Protein interaction studies involving recombinant Atp6v0e2 require careful experimental design to preserve native interactions and detect potentially transient associations:

• Bait protein preparation approaches:

  • Fusion tags: Use smaller tags (FLAG, HA) positioned at termini least likely to disrupt interactions based on structural data

  • Expression systems: Mammalian (HEK293T) or insect cells (Sf9) provide proper post-translational modifications

  • Solubilization conditions: Mild detergents (0.5-1% digitonin or 0.1-0.5% DDM) to preserve protein-protein interactions

• Interaction detection methods:

  • Co-immunoprecipitation: Optimized for membrane proteins with appropriate detergents

  • Proximity labeling: BioID or APEX2 fusion proteins to capture transient interactions

  • FRET/BRET assays: For monitoring interactions in living cells

  • Mammalian two-hybrid: For detecting interactions in a cellular context

• Crosslinking strategies:

  • Chemical crosslinkers (DSS, BS3) at low concentrations (0.5-2 mM)

  • Photo-crosslinking with incorporated photo-activatable amino acids

  • Crosslinking mass spectrometry (XL-MS) to identify specific interaction sites

• Validation approaches:

  • Reversed co-IP experiments with suspected interaction partners as bait

  • Competition experiments with excess untagged protein

  • Mutagenesis of predicted interaction interfaces based on structural data

  • In vitro binding assays with purified components

• Data analysis considerations:

  • Use appropriate negative controls (unrelated membrane proteins)

  • Validate hits with alternative interaction methods

  • Consider the stoichiometry of the V-ATPase complex when interpreting results

Recent structural studies have identified specific residues involved in Atp6v0e2 interactions (Arg77, Phe78, Leu79, Glu81) that can serve as targets for site-directed mutagenesis to validate and characterize specific protein-protein interactions within the V-ATPase complex .

What experimental approaches can distinguish the specific roles of Atp6v0e2 from other V-ATPase subunits?

Distinguishing the specific functions of Atp6v0e2 from other V-ATPase subunits requires sophisticated experimental approaches:

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated conditional knockout models specific for Atp6v0e2

  • Inducible shRNA or siRNA systems for temporal control of knockdown

  • Paralog-specific targeting to avoid affecting Atp6v0e1

  • Rescue experiments with wild-type versus mutant versions to identify critical functional domains

• Domain-specific functional analysis:

  • Structure-guided mutagenesis targeting the specific interaction residues identified in cryo-EM studies (Arg77, Phe78, Leu79, Glu81)

  • Chimeric constructs swapping domains between Atp6v0e2 and Atp6v0e1

  • Split-protein complementation assays to monitor specific interaction domains

• High-resolution imaging approaches:

  • Super-resolution microscopy (STORM, PALM) to visualize subunit-specific localization

  • Live-cell imaging with fluorescently tagged subunits to monitor dynamics

  • Correlative light and electron microscopy (CLEM) for structural context

  • FRET-based sensors to detect conformational changes in V-ATPase complexes

• Biochemical fractionation techniques:

  • Gradient centrifugation to separate assembled complexes from free subunits

  • Blue native PAGE to preserve and analyze intact complexes

  • Selective solubilization protocols to distinguish membrane-associated from fully integrated subunits

  • Pulse-chase experiments to monitor assembly kinetics

• Omics-based approaches:

  • Proteomics analysis of Atp6v0e2 interactome compared to other subunits

  • Transcriptomics to identify gene expression changes specific to Atp6v0e2 manipulation

  • Metabolomics to detect changes in lysosomal function indicators

Recent research utilizing transcriptome sequencing revealed that ATP6V0E2 is specifically upregulated in response to anlotinib treatment in cancer cells, suggesting distinct regulatory mechanisms and functions compared to other V-ATPase subunits . This type of differential response to experimental conditions provides valuable opportunities to distinguish subunit-specific roles .

How does Atp6v0e2 dysfunction contribute to disease pathogenesis in animal models?

While specific studies on Atp6v0e2 dysfunction in disease models are still emerging, research on V-ATPase components broadly suggests several pathogenic mechanisms:

Future research employing tissue-specific conditional knockout models for Atp6v0e2 will be valuable in elucidating its specific contributions to disease pathogenesis across different organ systems .

What experimental evidence links Atp6v0e2 to cancer progression and therapeutic response?

Recent research has uncovered significant connections between ATP6V0E2 and cancer biology, particularly in the context of therapeutic response:

• Transcriptional regulation in cancer treatment:

  • Transcriptome sequencing analysis revealed that ATP6V0E2 is significantly upregulated in human colon cancer cells treated with anlotinib, a receptor tyrosine kinase inhibitor .

  • This transcriptional response appears to be specific, as ATP6V0E2 was identified among a select group of differentially expressed genes following treatment .

• Lysosomal function modulation:

  • ATP6V0E2 upregulation coincided with enhanced lysosomal function and increased autophagy in cancer cells .

  • These processes were demonstrated through:

    • Increased LysoTracker staining indicating enhanced lysosomal acidification

    • Elevated expression of lysosomal enzymes

    • Enhanced autophagic flux measured via LC3-II/LC3-I ratios

    • Increased fusion of autophagosomes and lysosomes

• Mechanistic pathway identification:

  • Anlotinib treatment inhibits mTOR signaling, leading to TFEB activation and nuclear translocation .

  • TFEB serves as a master transcriptional regulator of lysosomal genes, including ATP6V0E2 .

  • Knockdown of either TFEB or ATP6V0E2 attenuated anlotinib-induced lysosomal activation, confirming this regulatory pathway .

• Therapeutic implications:

  • Paradoxically, inhibition of lysosomal function using bafilomycin A1 (a V-ATPase inhibitor) enhanced anlotinib-induced cell death and tumor suppression .

  • This suggests a dual role for ATP6V0E2 in cancer:

    • Initially upregulated as a protective response to therapy

    • Potentially contributing to treatment resistance through enhanced lysosomal function

    • Becoming a vulnerability when targeted alongside primary anticancer agents

These findings suggest that ATP6V0E2 status could serve as a potential biomarker for treatment response and that targeting V-ATPase function in combination with existing therapies might represent a promising strategy for enhancing cancer treatment efficacy .

How can researchers design experiments to identify potential therapeutic applications targeting Atp6v0e2?

Designing experiments to explore Atp6v0e2 as a therapeutic target requires a systematic approach spanning from basic mechanistic studies to preclinical evaluations:

• Target validation studies:

  • Gene expression manipulation: Employ CRISPR/Cas9, shRNA, or siRNA approaches to modulate Atp6v0e2 levels in disease models.

  • Rescue experiments: Reintroduce wild-type or mutant Atp6v0e2 to determine which domains/functions are critical.

  • Tissue-specific conditional knockout models: Generate models that allow temporal and spatial control of Atp6v0e2 deletion.

• High-throughput screening approaches:

  • Small molecule library screening: Develop cell-based assays that report on Atp6v0e2 function or V-ATPase activity.

  • Assay design considerations:

    • pH-sensitive fluorescent reporters to measure lysosomal acidification

    • Reporter cell lines expressing Atp6v0e2-fluorescent protein fusions

    • Phenotypic screens measuring endpoints like autophagy flux or lysosomal enzyme activity

  • Specificity counter-screens: Include assays to distinguish Atp6v0e2-specific from general V-ATPase effects.

• Combination therapy evaluations:

  • Based on findings that lysosomal inhibition enhances anlotinib efficacy in colon cancer , design experiments testing:

    • V-ATPase inhibitors (bafilomycin A1, concanamycin A) in combination with standard therapeutics

    • Specific Atp6v0e2 inhibition (when tools become available) versus pan-V-ATPase inhibition

    • Sequence and timing optimization for combination approaches

• Biomarker development studies:

  • Expression correlation analysis: Determine if Atp6v0e2 expression levels correlate with clinical outcomes.

  • Post-translational modification mapping: Identify regulatory modifications that might serve as measurable biomarkers.

  • Liquid biopsy approaches: Explore detection of Atp6v0e2 or its modifications in circulating tumor cells or exosomes.

• Preclinical model testing:

  • Cancer xenograft models: Test the impact of Atp6v0e2 modulation on tumor growth and response to therapy.

  • Patient-derived organoids: Evaluate effects in more physiologically relevant models.

  • Efficacy and toxicity assessment: Determine therapeutic window by comparing effects in disease versus normal tissues.

The research showing that ATP6V0E2 upregulation occurs in response to anlotinib treatment, and that inhibiting lysosomal function enhances therapeutic efficacy, provides a strong rationale for developing combination approaches targeting V-ATPase function alongside existing cancer therapies .

What are the most promising approaches for studying the structure-function relationship of Atp6v0e2?

Understanding the structure-function relationship of Atp6v0e2 requires integrating cutting-edge structural biology techniques with functional studies:

• Advanced structural biology approaches:

  • Cryo-electron microscopy: Recent advances have enabled visualization of V-ATPase complexes at near-atomic resolution, revealing specific interactions involving subunit e2 .

  • Integrative structural biology: Combining cryo-EM with cross-linking mass spectrometry (XL-MS) and molecular dynamics simulations to reveal dynamic aspects of Atp6v0e2 function.

  • Time-resolved structural studies: Capturing structural changes during the V-ATPase catalytic cycle to understand how Atp6v0e2 may contribute to conformational changes.

• Structure-guided mutagenesis:

  • Interaction interface targeting: Create mutations in the specific residues identified as forming interactions with other subunits (Arg77, Phe78, Leu79, Glu81) .

  • Systematic alanine scanning: Identify additional functionally important residues throughout the protein.

  • Domain swapping experiments: Create chimeric constructs between Atp6v0e2 and its paralog Atp6v0e1 to identify functional domains.

• Single-molecule approaches:

  • FRET-based sensors: Develop constructs with strategically placed fluorophores to monitor conformational changes during V-ATPase operation.

  • Optical tweezers or magnetic tweezers: Measure mechanical forces and energy transduction in reconstituted systems.

  • High-speed AFM: Visualize structural dynamics of V-ATPase complexes containing Atp6v0e2.

• In silico molecular dynamics:

  • Molecular dynamics simulations: Model the behavior of Atp6v0e2 within the V-ATPase complex in a lipid bilayer environment.

  • Free energy calculations: Quantify the energetic contribution of specific interactions involving Atp6v0e2.

  • Virtual screening: Identify potential small-molecule binding sites that could modulate Atp6v0e2 function.

• Functional correlation studies:

  • Structure-activity relationship (SAR) analysis: Correlate structural changes from mutagenesis with functional outcomes measured through proton pumping assays.

  • Protein stability assessment: Determine how mutations affect the assembly and stability of the V-ATPase complex.

  • In vivo rescue experiments: Test the ability of structure-guided mutants to rescue phenotypes in Atp6v0e2-deficient models.

Recent high-resolution structural studies have begun to reveal the specific interactions formed by subunit e2 with other V-ATPase components, providing a foundation for detailed structure-function investigations .

How might emerging technologies enhance our understanding of Atp6v0e2 regulation and function?

Emerging technologies offer unprecedented opportunities to elucidate Atp6v0e2 biology:

• Spatial transcriptomics and proteomics:

  • Single-cell spatial transcriptomics: Map Atp6v0e2 expression patterns with subcellular resolution across tissues.

  • Proximity proteomics: Techniques like BioID, APEX, or TurboID can identify proteins in close proximity to Atp6v0e2 in different subcellular compartments.

  • Spatial proteomics: CODEX or imaging mass cytometry to visualize Atp6v0e2 distribution alongside other markers.

• Advanced genome editing approaches:

  • Base editing and prime editing: Create precise point mutations in endogenous Atp6v0e2 without double-strand breaks.

  • CRISPRi/CRISPRa systems: Modulate Atp6v0e2 expression without altering the genomic sequence.

  • Knock-in reporter systems: Tag endogenous Atp6v0e2 with fluorescent proteins or epitope tags using precise editing.

• Live-cell imaging innovations:

  • Lattice light-sheet microscopy: Capture Atp6v0e2 dynamics with high spatiotemporal resolution and minimal phototoxicity.

  • Super-resolution microscopy: Techniques like STORM, PALM, or MINFLUX to visualize Atp6v0e2 localization beyond the diffraction limit.

  • Fluorescent biosensors: Develop sensors for V-ATPase activity or conformational changes to monitor function in real-time.

• Artificial intelligence and computational approaches:

  • Deep learning for image analysis: Enhance detection of subtle phenotypes in Atp6v0e2 manipulation experiments.

  • AlphaFold2 and RoseTTAFold: Predict structures of Atp6v0e2 variants or complexes not yet solved experimentally.

  • Network analysis: Integrate multi-omics data to place Atp6v0e2 in broader cellular regulatory networks.

• Organoid and microphysiological systems:

  • Tissue-specific organoids: Study Atp6v0e2 function in physiologically relevant 3D culture systems.

  • Organ-on-chip platforms: Examine Atp6v0e2 roles in complex tissue microenvironments with controlled parameters.

  • Patient-derived models: Correlate Atp6v0e2 function with patient-specific disease phenotypes.

Recent research has employed transcriptome sequencing to identify ATP6V0E2 as a key gene upregulated in response to anlotinib treatment, demonstrating how -omics approaches can reveal unexpected regulatory relationships . Integration of these technologies promises to provide a comprehensive understanding of Atp6v0e2 regulation and function in both normal physiology and disease states.

What are the key unresolved questions about Atp6v0e2 that warrant further investigation?

Despite recent advances, several critical questions about Atp6v0e2 remain unanswered and represent important areas for future research:

• Evolutionary and paralog-specific functions:

  • Why have two e subunit paralogs (e1 and e2) been maintained throughout evolution?

  • What are the specific functional differences between Atp6v0e1 and Atp6v0e2?

  • Are there tissue-specific or condition-specific requirements for Atp6v0e2 versus Atp6v0e1?

• Regulatory mechanisms:

  • How is Atp6v0e2 expression regulated at the transcriptional and post-transcriptional levels?

  • What signaling pathways beyond TFEB-mediated transcription control Atp6v0e2 expression?

  • Does Atp6v0e2 undergo post-translational modifications that regulate its function?

• Structural contributions:

  • How does Atp6v0e2 contribute to the stability and/or assembly of the V-ATPase complex?

  • Does Atp6v0e2 play a role in the reversible dissociation of V1 and V0 domains used for regulation?

  • Could Atp6v0e2 influence the coupling efficiency between ATP hydrolysis and proton pumping?

• Disease relevance:

  • Are there human diseases specifically associated with ATP6V0E2 mutations or dysregulation?

  • How does ATP6V0E2 expression correlate with cancer progression or treatment resistance?

  • Could tissue-specific functions of ATP6V0E2 explain differential sensitivity of tissues to V-ATPase inhibitors?

• Therapeutic targeting:

  • Is it possible to develop inhibitors specific to ATP6V0E2 versus other V-ATPase subunits?

  • Would ATP6V0E2-specific targeting provide advantages over general V-ATPase inhibition?

  • Could ATP6V0E2 status serve as a biomarker for predicting response to therapies targeting lysosomal function?