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

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Description

Overview of Recombinant Human V-type Proton ATPase Subunit e2 (ATP6V0E2)

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 .

Key Features

PropertyDetails
Gene IDENSG00000171130 (human)
UniProt IDQ8NHE4 (human)
Subunit RoleMembrane-embedded V0 subunit; stabilizes proton translocation
Tissue ExpressionUbiquitous; high in adrenal glands, bone marrow, and colon
Disease AssociationImplicated in osteopetrosis and cancer progression

Role in Lysosomal Function

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) .

StudyKey Findings
Anlotinib MechanismATP6V0E2 knockdown reduces lysosomal activation, increasing anlotinib cytotoxicity
TFEB RegulationAnlotinib promotes TFEB nuclear translocation, enhancing ATP6V0E2 transcription
Cancer SubtypingATP6V0E2 expression correlates with distinct immunological tumor subtypes in colorectal cancer

Targeted Cancer Therapy

  • 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 .

Genetic Variations

Mutations in ATP6V0E2 have been identified in cancer cohorts, though their functional impact remains under investigation .

Gene and Protein Details

ParameterValue
Chromosomal Location7q31.1 (human)
Transcript Length1,287 bp (NM_001283978)
Protein Length247 amino acids
ParalogsATP6V0E1 (isoform-specific tissue distribution)

Product Specs

Form
Lyophilized powder
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized preparation.
Lead Time
Delivery times vary depending on the purchase method and location. Please contact your local distributor for precise delivery estimates.
Note: All proteins are shipped with standard blue ice packs unless dry ice is specifically requested in advance. Additional fees apply for dry ice shipping.
Notes
Avoid repeated freeze-thaw cycles. Store working aliquots at 4°C for up to one week.
Reconstitution
Centrifuge the vial briefly before opening to consolidate the contents. Reconstitute the protein in sterile deionized water to a concentration of 0.1-1.0 mg/mL. For long-term storage, we recommend adding 5-50% glycerol (final concentration) and aliquoting at -20°C/-80°C. Our standard glycerol concentration is 50% and serves as a guideline.
Shelf Life
Shelf life depends on various factors including storage conditions, buffer composition, temperature, and protein stability. Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquot to prevent repeated freeze-thaw cycles.
Tag Info
Tag type is determined during manufacturing.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Synonyms
ATP6V0E2; ATP6V0E2L; C7orf32; V-type proton ATPase subunit e 2; V-ATPase subunit e 2; Lysosomal 9 kDa H(+-transporting ATPase V0 subunit e2; Vacuolar proton pump subunit e 2
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-81
Protein Length
full length protein
Species
Homo sapiens (Human)
Target Names
Target Protein Sequence
MTAHSFALPVIIFTTFWGLVGIAGPWFVPKGPNRGVIITMLVATAVCCYLFWLIAILAQL NPLFGPQLKNETIWYVRFLWE
Uniprot No.

Target Background

Function

Vacuolar ATPases are responsible for acidifying various intracellular compartments in eukaryotic cells.

Gene References Into Functions
  1. The identification of this novel e-subunit form further supports the hypothesis that subunit variations play a crucial role in the structure, localization, and function of H+-ATPases within the cell. [ATPV0E2] PMID: 17350184
Database Links

HGNC: 21723

OMIM: 611019

KEGG: hsa:155066

UniGene: Hs.556998

Protein Families
V-ATPase e1/e2 subunit family
Subcellular Location
Membrane; Multi-pass membrane protein.
Tissue Specificity
Isoform 1 is expressed at high levels in heart, brain and kidney and also detected in inner ear epithelium, vestibule, testis, epididymis and bladder. Isoform 2 is expressed in heart, kidney, placenta and pancreas. Isoform 2 is not detected in frontal cor

Q&A

What is ATP6V0E2 and what is its role in V-ATPase function?

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 .

What cellular processes involve ATP6V0E2?

Research indicates that ATP6V0E2 participates in multiple cellular processes:

  • Acidification of intracellular vesicles and organelles

  • Lysosomal function and autophagy regulation

  • Autophagosome-lysosome fusion processes

  • Insulin receptor recycling pathways

  • Cellular responses to external stimuli

  • 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 .

What diseases are associated with ATP6V0E2 dysfunction?

ATP6V0E2 dysfunction has been linked to several pathologies:

DiseaseMechanismEvidence
OsteopetrosisImpaired bone resorption due to defective proton transport in osteoclastsGene association studies
Colon cancerAltered lysosomal function affecting treatment response and cell survivalTranscriptome sequencing showing upregulation with anlotinib treatment
Other cancersChanges in tumor microenvironment pH and autophagy regulationModulation of lysosomal function in cancer cells

The involvement of ATP6V0E2 in these diseases highlights its potential as a therapeutic target, particularly in combination therapy approaches for cancer .

What is the role of ATP6V0E2 in cancer progression and treatment response?

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 .

How does ATP6V0E2 contribute to V-ATPase assembly and biogenesis?

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 .

How do post-translational modifications affect ATP6V0E2 function?

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 .

What experimental approaches are optimal for studying ATP6V0E2 knockdown effects?

For investigating ATP6V0E2 function through knockdown experiments, several validated methodologies have proven effective:

ApproachMethodologyKey ParametersApplication
siRNA transfectionTransfect using Lipofectamine® 300048-hour post-transfection analysisValidated in HCT116 colon cancer cells
Colony formation assaySeed 200 cells in 6-well plates, treat with anlotinib, culture 12-20 daysCount colonies ≥50 cells after gentian violet stainingAssess long-term effects of ATP6V0E2 knockdown on cell survival
Western blottingProtein extraction, SDS-PAGE, immunoblottingAnalyze cleaved PARP-1, Caspase-3, Bax, Cytochrome c, Bcl-2Evaluate apoptotic pathway activation
Transcriptome sequencingRNA extraction, library preparation, 150-bp paired-end sequencingIllumina X Ten platformIdentify differentially expressed genes following knockdown
TFEB luciferase assayCo-transfect TFEB luciferase vector and Renilla controlMeasure luciferase activity at 48 hours post-transfectionAssess transcriptional regulation mechanisms

When designing ATP6V0E2 knockdown experiments, researchers should consider including appropriate controls and validating knockdown efficiency through both mRNA and protein level measurements .

How can researchers effectively generate recombinant ATP6V0E2 for structural studies?

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 .

What techniques are most effective for studying ATP6V0E2 localization and interactions?

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.

How can researchers quantify ATP6V0E2-mediated proton transport activity?

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.

What are the key unanswered questions about ATP6V0E2 function?

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?

How might ATP6V0E2 research impact cancer treatment strategies?

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.

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