ATP6V0E2 is a critical subunit of the V0 domain in vacuolar-type H⁺-ATPases (V-ATPases), responsible for proton transport across cellular membranes. Recombinant ATP6V0E2 is engineered for research or therapeutic applications, enabling precise study of its role in acidifying intracellular compartments (e.g., lysosomes, endosomes) and plasma membrane proton extrusion .
ATP6V0E2 is upregulated by anlotinib, a tyrosine kinase inhibitor used in colorectal cancer therapy. This upregulation enhances lysosomal acidification and autophagy, which counteracts anlotinib-induced apoptosis by reducing reactive oxygen species (ROS) .
Lysosomal Inhibition: Co-treatment with lysosomal inhibitors (e.g., chloroquine) enhances anlotinib efficacy by disrupting ATP6V0E2-mediated proton transport, leading to ROS accumulation and apoptosis .
Biomarker Potential: ATP6V0E2 expression levels may predict responsiveness to V-ATPase-targeted therapies in solid tumors .
Mutations in ATP6V0E2 have been identified in cancer cohorts, though their functional impact remains under investigation .
Vacuolar ATPases are responsible for acidifying various intracellular compartments in eukaryotic cells.
ATP6V0E2 is a subunit of the V0 complex of vacuolar-type H+-ATPase. V-ATPases are ATP-driven proton pumps composed of two main complexes: the cytoplasmic V1 complex that hydrolyzes ATP, and the membrane-embedded V0 complex responsible for proton transfer across membranes . As part of the integral membrane domain, ATP6V0E2 contributes to the proton translocation pathway that is essential for acidifying various intracellular compartments including vacuoles, lysosomes, endosomes, and other organelles . The protein functions within a coordinated mechanical assembly where ATP hydrolysis by V1 drives rotation of the V0 ring to facilitate proton transport .
Research indicates that ATP6V0E2 participates in multiple cellular processes:
Bone resorption (related to its association with osteopetrosis)
Cancer cell metabolism and response to targeted therapies like anlotinib
Recent transcriptome profiling revealed that ATP6V0E2 expression changes in response to anti-cancer treatments, suggesting a role in treatment efficacy and resistance mechanisms .
ATP6V0E2 dysfunction has been linked to several pathologies:
The involvement of ATP6V0E2 in these diseases highlights its potential as a therapeutic target, particularly in combination therapy approaches for cancer .
ATP6V0E2 appears to play a critical role in cancer cell response to targeted therapies. Transcriptome profiling analysis revealed that anlotinib, a receptor tyrosine kinase inhibitor used in cancer treatment, significantly upregulates ATP6V0E2 and other lysosome-related genes in human colon cancer cells . This upregulation correlates with enhanced lysosomal function and increased autophagosome-lysosome fusion .
The functional significance of this upregulation is complex:
Increased lysosomal function appears to protect against anlotinib-mediated cell apoptosis by regulating cellular redox status
When ATP6V0E2 is knocked down, the enhanced lysosomal function and autophagy induced by anlotinib are attenuated
Inhibition of lysosomal function enhances anlotinib-induced cell death and tumor suppression
These findings suggest that ATP6V0E2-mediated lysosomal activation serves as a protective mechanism against anti-cancer treatments, and targeting this pathway could enhance therapeutic efficacy .
Recent cryo-electron microscopy structures of human V-ATPase at up to 2.9 Å resolution have provided insights into V-ATPase assembly mechanisms . While specific details about ATP6V0E2's assembly role are still emerging, several key observations are relevant:
V-ATPase assembly requires coordinated incorporation of protein subunits, glycans, and lipids
ATP6AP1 serves as a structural hub for V0 complex assembly, connecting multiple V0 subunits (potentially including ATP6V0E2) and phospholipids inside the c-ring
N-linked glycans, glycolipids, and phospholipids have been identified in the V0 complex and appear critical for proper assembly
The glycolipids and glycosylated V0 subunits form a luminal glycan coat essential for V-ATPase folding, localization, and stability
ATP6V0E2 likely participates in this assembly network, contributing to the structural integrity and functional capacity of the V0 complex through specific protein-protein and protein-lipid interactions .
Post-translational modifications, particularly glycosylation, appear critical for V-ATPase function. Cryo-EM studies combined with mass spectrometry have identified N-linked glycans associated with V0 subunits that form a luminal glycan coat essential for proper V-ATPase folding, localization, and stability .
The regulation of ATP6V0E2 also appears to involve the mTOR (mammalian target of rapamycin) signaling pathway and TFEB (transcription factor EB), a key transcriptional regulator of lysosomal biogenesis . Research indicates that:
Anlotinib inhibits mTOR signaling, which normally regulates TFEB through phosphorylation
TFEB activation promotes nuclear translocation and enhances its transcriptional activity on lysosomal genes
When TFEB is knocked down, the enhanced lysosomal function induced by anlotinib is attenuated, similar to the effect of ATP6V0E2 knockdown
This suggests a regulatory pathway where mTOR inhibition activates TFEB, which then upregulates ATP6V0E2 and other lysosomal genes to enhance lysosomal function .
For investigating ATP6V0E2 function through knockdown experiments, several validated methodologies have proven effective:
When designing ATP6V0E2 knockdown experiments, researchers should consider including appropriate controls and validating knockdown efficiency through both mRNA and protein level measurements .
Generating functional recombinant ATP6V0E2 for structural and biochemical studies requires careful consideration of expression systems and purification strategies:
Expression system selection:
Mammalian expression systems (HEK293 or CHO cells) are preferable for maintaining proper glycosylation patterns
Insect cell systems (Sf9 or Hi5) can provide high yields with some post-translational modifications
Bacterial systems may be suitable for non-glycosylated protein domains
Vector design considerations:
Include affinity tags (His6, FLAG, or Strep) for purification
Consider fusion proteins to enhance solubility
Include TEV or PreScission protease sites for tag removal
Codon optimization for the selected expression system
Purification strategy:
Membrane protein extraction using gentle detergents (DDM, LMNG, or digitonin)
Affinity chromatography as initial capture step
Size exclusion chromatography for final polishing
Consider lipid nanodisc or amphipol reconstitution for stability
Quality assessment:
SDS-PAGE and Western blot to confirm purity and identity
Mass spectrometry to verify protein sequence and modifications
Dynamic light scattering to assess homogeneity
Functional assays to confirm activity
For structural studies, cryo-electron microscopy has proven most effective for studying intact V-ATPase complexes, achieving resolutions of 2.9-3.0 Å for V1 and V0 subcomplexes respectively .
Several complementary approaches can provide insights into ATP6V0E2 localization and protein-protein interactions:
Subcellular localization:
Immunofluorescence microscopy with ATP6V0E2-specific antibodies
Co-localization studies with organelle markers (LAMP1 for lysosomes)
Live-cell imaging using fluorescent protein fusions (if function is preserved)
Subcellular fractionation followed by Western blotting
Protein-protein interaction studies:
Co-immunoprecipitation with ATP6V0E2-specific antibodies
Proximity labeling approaches (BioID or APEX2)
Crosslinking mass spectrometry to capture transient interactions
Fluorescence resonance energy transfer (FRET) for direct interaction partners
Structural interaction mapping:
Cryo-electron microscopy of intact V-ATPase complexes
Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces
Molecular modeling based on existing V-ATPase structures
Recent structural studies have revealed that ATP6AP1 acts as a structural hub for V0 complex assembly, connecting multiple V0 subunits and phospholipids . Investigating ATP6V0E2's interactions with ATP6AP1 and other V0 subunits could provide valuable insights into V-ATPase assembly mechanisms.
Measuring V-ATPase-mediated proton transport requires specialized techniques that can isolate the contribution of specific subunits like ATP6V0E2:
Acidification assays:
LysoTracker or LysoSensor dyes to measure lysosomal pH in intact cells
ACMA (9-amino-6-chloro-2-methoxyacridine) quenching assays with reconstituted proteoliposomes
Ratiometric pH measurements using pH-sensitive fluorescent proteins
ATP hydrolysis coupling:
ATPase activity assays (NADH-coupled or Pi release) in purified V-ATPase
Comparison between wild-type and ATP6V0E2-mutant complexes
Inhibitor sensitivity profiles (bafilomycin A1, concanamycin A)
Structure-function analysis:
Site-directed mutagenesis of key residues in ATP6V0E2
Rescue experiments in ATP6V0E2-depleted cells
Correlation between structural changes and transport activity
When ATP6V0E2 or TFEB are knocked down, lysosomal function and autophagy activation by anlotinib are attenuated , providing a functional readout of ATP6V0E2 activity in cellular contexts.
Despite recent advances, several fundamental questions about ATP6V0E2 remain unanswered:
What is the precise atomic structure of ATP6V0E2 within the V0 complex, and how does it contribute to proton translocation?
How do tissue-specific expression patterns of ATP6V0E2 contribute to specialized functions in different cell types?
What is the regulatory network controlling ATP6V0E2 expression, and how is it dysregulated in disease states?
How do specific post-translational modifications (glycosylation, phosphorylation) affect ATP6V0E2 function?
Can selective targeting of ATP6V0E2 provide therapeutic benefits without disrupting essential V-ATPase functions in normal cells?
Research on ATP6V0E2 has significant implications for cancer therapeutics:
Combination therapy development:
Research shows that inhibition of lysosomal function enhances anlotinib-induced cell death and tumor suppression . This suggests that combining V-ATPase inhibitors with receptor tyrosine kinase inhibitors could be a promising therapeutic strategy.
Biomarker potential:
ATP6V0E2 expression levels could potentially serve as a biomarker for predicting response to certain therapies, particularly those that affect autophagy and lysosomal function.
Resistance mechanism elucidation:
Understanding how ATP6V0E2-mediated lysosomal activation protects against anti-cancer treatments could help develop strategies to overcome treatment resistance.
Targeted delivery approaches:
Knowledge of ATP6V0E2's role in lysosomal function could inform the development of drug delivery systems that leverage lysosomal properties for enhanced therapeutic efficacy.
In human colon cancer cells, anlotinib treatment upregulates ATP6V0E2 and activates lysosomal function via inhibiting mTOR signaling and enhancing TFEB transcriptional activity . This activation of lysosomal function appears to protect against anlotinib-mediated cell apoptosis by regulating cellular redox status , suggesting that targeting this protective mechanism could enhance therapeutic efficacy.