TMEM127 Antibody

What is TMEM127 and why is it important in research?

TMEM127 is a tumor suppressor gene encoding a transmembrane protein that is frequently mutated in pheochromocytomas and, less commonly, in renal cancers. The significance of TMEM127 in research stems from its role in regulating mTORC1 signaling through interaction with the lysosome-anchored complex comprising Rag GTPases, the LAMTOR pentamer, and vATPase . This complex plays a crucial role in amino acid-mediated mTORC1 activation. Mutations in TMEM127 lead to increased mTORC1 signaling, contributing to tumorigenesis through mechanisms that researchers are actively investigating . Understanding TMEM127's function is essential for developing targeted therapies for cancers with TMEM127 mutations.

What applications are TMEM127 antibodies suitable for?

TMEM127 antibodies can be used in multiple experimental applications, including:

For optimal results, titration of the antibody is recommended for each experimental system .

How should researchers validate TMEM127 antibody specificity?

Validation of TMEM127 antibody specificity should include:

• Negative controls using TMEM127 knockout cells, as demonstrated in studies with HEK293T TMEM127-KO cells .

• Positive controls using tissues known to express TMEM127, such as heart tissue or specific cancer tissues .

• Peptide competition assays to confirm binding specificity.

• Multiple detection methods (e.g., Western blot and immunofluorescence) to confirm consistent results.

• Comparison with recombinant TMEM127 expression systems, particularly when studying specific variants.

When working with suspected TMEM127 mutations, researchers should compare antibody reactivity between wild-type and mutant TMEM127 to assess potential epitope alterations .

What are the optimal conditions for immunodetection of TMEM127?

For optimal immunodetection of TMEM127:

Immunohistochemistry (IHC):

• Antigen retrieval: Use TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

• Working dilution: 1:250-1:1000

• Positive control tissues: Human stomach cancer tissue, heart tissue, liver cancer tissue

Immunofluorescence:

• Fixation: 4% paraformaldehyde in PBS is suitable for most applications

• Permeabilization: Different permeabilization agents reveal different aspects of TMEM127 localization

  • Digitonin (mild detergent): Selectively permeabilizes plasma membrane

  • Triton X-100 (harsh detergent): Enables full permeabilization of all membranes

• Working dilution: 1:200-1:800

Immunoprecipitation:

• Lysate preparation: Use 1.0-3.0 mg of total protein lysate

• Antibody amount: 0.5-4.0 μg per IP reaction

How can researchers differentiate between various subcellular localizations of TMEM127?

TMEM127 localizes to multiple subcellular compartments, complicating its detection and analysis. To differentiate between various localizations:

• Co-localization studies: Use confocal microscopy with markers for:

  • Lysosomes (LAMP1, LAMP2)

  • Early endosomes

  • Late endosomes

  • Plasma membrane

• Membrane fractionation: Separate cytosolic and membrane-enriched fractions. TMEM127 is predominantly detected in membrane-enriched fractions containing lysosomes (LAMP2 positive) but not in cytosolic fractions .

• Time-course experiments: After amino acid stimulation, TMEM127 colocalization with lysosomal markers (LAMP2) increases, peaking at approximately 10 minutes before gradually decreasing . This dynamic profile resembles mTORC1 association with LAMP2.

• Permeabilization techniques: Use selective membrane permeabilization to distinguish between plasma membrane and internal membrane localization:

  • Without detergent: Only extracellularly facing epitopes are detected

  • With digitonin: Plasma membrane is selectively permeabilized

  • With Triton X-100: All membranes are permeabilized

What methodological approaches help determine TMEM127 membrane topology?

Determining TMEM127 membrane topology requires multiple complementary approaches:

• Antibody accessibility assays: Using antibodies directed against different termini of TMEM127 under various permeabilization conditions. Research has shown that both N- and C-termini of TMEM127 are oriented toward the cytoplasm, supporting an even number of transmembrane domains .

• Computational prediction tools: Hydrophobicity analyses and transmembrane domain prediction algorithms can identify potential membrane-spanning regions. These predictions should be experimentally validated .

• Terminal tagging strategies: Creating constructs with epitope tags at either terminus allows for orientation determination using selective permeabilization.

• Protease protection assays: Regions exposed to the cytoplasm are susceptible to protease digestion, while lumenal domains are protected.

Recent research has revealed that TMEM127 contains four transmembrane domains, contradicting earlier models suggesting only three domains. A previously unrecognized transmembrane domain was identified between residues 30 and 53 .

How can researchers analyze TMEM127 interaction with the mTORC1 signaling pathway?

To analyze TMEM127 interaction with mTORC1 signaling:

• Co-immunoprecipitation studies:

  • TMEM127 interacts with LAMTOR proteins and can be detected by immunoprecipitated recombinant LAMTOR1

  • HA-TMEM127 immunoprecipitates endogenous LAMTOR1

  • Validation through IP of endogenous proteins is essential

• Amino acid stimulation experiments:

  • Starve cells of amino acids (typically 50-60 minutes)

  • Stimulate with amino acids for various time points (5, 10, 20, 30 minutes)

  • Analyze phosphorylation of mTORC1 downstream targets (pS6K, pS6)

  • Compare TMEM127 wild-type vs. knockout or knockdown conditions

• Rag GTPase binding assays:

  • Express TMEM127 in cells carrying HA-GST-RagD

  • Assess binding between RagD and endogenous mTOR and Raptor

  • Test effects of constitutively active (RagB Q99L + RagD S77L) and inactive (RagB T54L + RagD Q121L) Rag heterodimers

• Lysosomal recruitment analysis:

  • Use confocal microscopy to quantify mTOR/LAMP1 colocalization under various amino acid conditions

  • Compare TMEM127-null cells with those re-expressing TMEM127

What methods are effective for studying TMEM127 variants and mutations?

Functional characterization of TMEM127 variants requires multiple methodological approaches:

• Subcellular localization analysis:

  • Express tagged TMEM127 variants in TMEM127-knockout cells

  • Use immunofluorescence to compare localization patterns

  • Wild-type TMEM127 shows punctate endomembrane distribution

  • Mutant constructs often display diffuse cytoplasmic distribution

• Protein stability assessment:

  • Measure steady-state levels of TMEM127 variants by immunoblot

  • Conduct cycloheximide chase experiments to determine protein half-life

  • Compare degradation rates between wild-type and mutant proteins

• Membrane association studies:

  • Perform membrane fractionation experiments

  • Determine whether variants retain ability to associate with membranes

  • Complement with microscopy to identify specific membrane compartments

• Functional rescue experiments:

  • Re-express TMEM127 variants in TMEM127-null cells

  • Measure rescue of phenotypes such as mTORC1 hyperactivation

  • Assess correction of LAMTOR protein accumulation

Recent studies have identified three subgroups of mutations and determined that approximately 71% of tumor-associated variants, including 60% of missense variants, are pathogenic or likely pathogenic due to loss of membrane binding ability, stability, and/or internalization capability .

How can TMEM127 antibodies be used to study endocytic trafficking mechanisms?

TMEM127 undergoes internalization through clathrin-mediated endocytosis, providing a model for studying this process:

• Internalization assays:

  • Label cell surface TMEM127 using antibodies against extracellular domains or epitope tags

  • Allow internalization at 37°C for various time points

  • Remove remaining surface label and quantify internalized protein

• Mutational analysis of trafficking motifs:

  • C-terminal variants with predominant plasma membrane localization reveal an atypical, extended acidic, dileucine-based motif required for TMEM127 internalization

  • Create point mutations in this motif to assess effects on endocytosis

• Colocalization with endocytic markers:

  • Track TMEM127 progression through early endosomes, late endosomes, and lysosomes

  • Use markers such as EEA1 (early endosomes), Rab7 (late endosomes), and LAMP1/2 (lysosomes)

• Endocytic pathway inhibitors:

  • Use chlorpromazine or dynasore to inhibit clathrin-mediated endocytosis

  • Apply filipin or methyl-β-cyclodextrin to disrupt caveolae-mediated endocytosis

  • Determine which pathway predominantly regulates TMEM127 trafficking

How should researchers interpret conflicting TMEM127 subcellular localization data?

When facing conflicting TMEM127 localization data:

• Consider amino acid status: TMEM127 localization is dynamic and dependent on amino acid availability. Colocalization with lysosomal markers increases after amino acid exposure, peaking around 10 minutes before gradually decreasing .

• Evaluate expression levels: Overexpression can lead to saturation of trafficking machinery and mislocalization. Compare endogenous localization with various expression levels of recombinant protein.

• Cell type variations: TMEM127 exhibits ubiquitous expression but may show cell type-specific localization patterns. Compare findings across multiple cell lines.

• Antibody epitope accessibility: TMEM127's complex membrane topology may result in epitope masking in certain cellular compartments. Use multiple antibodies targeting different regions of the protein.

• Fixation and permeabilization effects: Different methods can selectively preserve or expose certain localizations:

  • Paraformaldehyde fixation preserves membrane structures

  • Methanol fixation can disrupt membranes

  • Various detergents (Triton X-100, digitonin, saponin) selectively permeabilize different membrane compartments

What are the key considerations when analyzing TMEM127-null tumors or cell models?

When analyzing TMEM127-null tumors or cell models:

• Validation of TMEM127 loss: Confirm complete absence of TMEM127 expression using multiple methods (Western blot, qPCR, immunostaining).

• Expected molecular changes:

  • Increased levels of LAMTOR1 and LAMTOR2 proteins compared to wild-type TMEM127 samples

  • Potential increases in Rag and vATPase protein levels

  • Enhanced activation of mTORC1 downstream targets (pS6K and pS6) upon amino acid stimulation

• Functional consequences:

  • Increased mTORC1 recruitment to lysosomes under amino acid-rich conditions

  • Enhanced sensitivity to mTORC1 inhibitors like rapamycin

  • Altered nutrient sensing responses

• Compensatory mechanisms: Long-term TMEM127 loss may activate compensatory pathways that mask acute effects.

• Genetic background considerations: The impact of TMEM127 loss may vary depending on the status of other genes in the mTORC1 pathway.

What are the common pitfalls in TMEM127 immunoprecipitation experiments?

Common pitfalls in TMEM127 immunoprecipitation experiments include:

• Epitope masking: TMEM127's membrane association and protein interactions may obscure antibody binding sites. Test multiple antibodies targeting different regions.

• Cross-reactivity: Validate antibody specificity using TMEM127-knockout controls to ensure signals are specific .

• Detergent selection: Membrane protein extraction requires careful detergent selection:

  • Too harsh: May disrupt protein-protein interactions

  • Too mild: May fail to solubilize membrane-bound TMEM127

  • Recommended: CHAPS, NP-40, or Triton X-100 at optimized concentrations

• Transient interactions: Some TMEM127 interactions (e.g., with LAMTOR components) are dynamic and amino acid-dependent . Consider crosslinking or stimulus-specific timing.

• Low expression levels: Endogenous TMEM127 is often expressed at low levels. Scale up input material or use enrichment strategies.

• Sample processing: Rapid processing is essential as TMEM127-LAMTOR interactions may be unstable during prolonged handling.

How can TMEM127 antibodies contribute to understanding pheochromocytoma pathogenesis?

TMEM127 antibodies can advance understanding of pheochromocytoma pathogenesis through:

• Tumor sample analysis:

  • Primary pheochromocytomas carrying germline truncating TMEM127 mutations show loss of the wild-type allele and no residual TMEM127 expression

  • These tumors exhibit consistently higher levels of LAMTOR1 and LAMTOR2 proteins compared to pheochromocytomas with wild-type TMEM127

  • Trends toward increased Rag and vATPase protein levels suggest broader effects on lysosomal scaffold components

• Diagnostic applications:

  • Immunohistochemical screening of tumor samples for TMEM127 expression

  • Correlation of TMEM127 loss with mTORC1 pathway activation markers

  • Identification of potential therapeutic vulnerabilities

• Mechanistic insights:

  • Analysis of mTORC1 signaling components in TMEM127-mutant vs. wild-type tumors

  • Examination of lysosomal function and nutrient sensing in tumor samples

  • Investigation of potential metabolic adaptations associated with TMEM127 loss

• Comparative studies:

  • Compare TMEM127-mutant pheochromocytomas with other genetic subtypes

  • Assess overlap with tumors carrying mutations in other mTORC1 pathway components

  • Identify molecular subgroups that might benefit from specific therapeutic strategies

What methodological approaches help determine the functional impact of novel TMEM127 variants?

To determine the functional impact of novel TMEM127 variants:

• Structure-function classification:

  • Group variants based on their effects on protein localization, stability, and function

  • Recent research has identified three subgroups of mutations based on functional deficits

• Multi-tiered experimental assessment:

  • Subcellular localization analysis using fluorescence microscopy

  • Protein stability evaluation through immunoblotting

  • Membrane topology confirmation using antibody accessibility assays

  • Endocytic capability measurement through internalization assays

• Computational prediction integration:

  • In silico analysis of potential transmembrane domains

  • Conservation assessment across species

  • Structural modeling of variant effects on protein folding

  • Integration with experimental data for comprehensive classification

• Functional complementation:

  • Expression of variants in TMEM127-null backgrounds

  • Assessment of rescue of characteristic phenotypes:

    • mTORC1 hyperactivation

    • LAMTOR protein accumulation

    • Altered amino acid sensing

This multi-faceted approach has successfully classified 71% of tumor-associated variants, including 60% of missense variants, as pathogenic or likely pathogenic through loss of membrane binding ability, stability, and/or internalization capability .

How should researchers interpret TMEM127 staining patterns in different tumor types?

When interpreting TMEM127 staining patterns in tumor samples:

• Expression level assessment:

  • Complete loss: Consistent with biallelic inactivation, often seen in pheochromocytomas with germline TMEM127 mutations and loss of heterozygosity

  • Reduced expression: May indicate monoallelic loss or downregulation

  • Normal/increased expression: May suggest alternative pathogenic mechanisms

• Subcellular distribution patterns:

  • Punctate endomembrane staining: Typical of wild-type TMEM127

  • Diffuse cytoplasmic pattern: Often seen with truncating or missense mutations that disrupt membrane binding

  • Plasma membrane accumulation: May indicate defects in internalization motifs

• Correlation with downstream markers:

  • LAMTOR protein levels: Typically elevated in TMEM127-null tumors

  • mTORC1 activation markers (pS6K, pS6): Often increased

  • Lysosomal distribution: May be altered

• Comparative analysis:

  • Compare patterns across different tumor types (pheochromocytoma, paraganglioma, renal carcinoma)

  • Assess correlation with genetic data when available

  • Consider tissue-specific differences in TMEM127 expression and localization

• Controls and validation:

  • Include known TMEM127-mutant samples as references

  • Use multiple antibodies targeting different epitopes

  • Perform genetic analysis when unusual patterns are observed

How might antibodies against TMEM127 be used to study therapeutic targeting of the mTORC1 pathway?

Antibodies against TMEM127 can facilitate research into therapeutic targeting of the mTORC1 pathway through:

• Biomarker development:

  • Identification of patient populations with TMEM127 alterations

  • Correlation of TMEM127 status with response to mTORC1 inhibitors

  • Detection of compensatory mechanisms during treatment

• Mechanistic studies:

  • Investigation of TMEM127's precise role in regulating mTORC1 at the lysosome

  • Analysis of how TMEM127 affects LAMTOR/ragulator stability and function

  • Exploration of potential differential effects on specific mTORC1 substrates

• Drug development support:

  • Screening for compounds that modulate TMEM127-dependent mTORC1 regulation

  • Evaluation of combination therapies targeting both TMEM127 and mTORC1 pathways

  • Assessment of specificity for TMEM127-deficient vs. wild-type cells

• Therapeutic resistance mechanisms:

  • Study of adaptive responses to mTORC1 inhibition in TMEM127-deficient contexts

  • Identification of bypass pathways that emerge during treatment

  • Development of strategies to overcome resistance

What experimental approaches can resolve contradictions in TMEM127 membrane topology models?

To resolve contradictions in TMEM127 membrane topology models:

• Integrated structural biology approaches:

  • Cryogenic electron microscopy of purified TMEM127

  • X-ray crystallography of individual domains or full-length protein

  • NMR spectroscopy of specific transmembrane segments

• Systematic mutagenesis:

  • Creation of a comprehensive panel of mutations throughout the protein

  • Assessment of each mutation's effect on membrane integration

  • Identification of critical residues for membrane topology

• Advanced topology mapping:

  • APEX2 proximity labeling at various positions within TMEM127

  • Glycosylation site insertion analysis

  • Cysteine accessibility methods

• Computational prediction refinement:

  • Integration of machine learning approaches with experimental data

  • Molecular dynamics simulations of membrane insertion and folding

  • Evolutionary analysis of conservation patterns in transmembrane regions

Recent research has challenged the previous three-transmembrane domain model, providing evidence for a four-transmembrane domain structure with both N- and C-termini oriented toward the cytoplasm . The newly identified transmembrane domain is located between residues 30 and 53 .

How can researchers effectively analyze the dynamics of TMEM127-LAMTOR interactions?

To effectively analyze TMEM127-LAMTOR interaction dynamics:

• Live-cell imaging approaches:

  • Fluorescently tagged TMEM127 and LAMTOR components

  • FRET or BRET assays to monitor protein-protein interactions in real-time

  • Photobleaching techniques (FRAP, FLIP) to assess mobility and exchange rates

• Stimulus-dependent interaction analysis:

  • Time-course experiments following amino acid stimulation

  • Selective inhibition of specific signaling components

  • Comparison between wild-type and mutant proteins

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify neighboring proteins

  • Time-resolved proximity labeling to capture dynamic interactions

  • Comparison of interactomes under different nutrient conditions

• Structural characterization:

  • Cryo-EM analysis of the TMEM127-LAMTOR complex

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cross-linking mass spectrometry to identify contact points

• Domain mapping:

  • Expression of truncation mutants to identify interaction domains

  • Point mutations at conserved residues

  • Competition assays with peptides derived from interaction interfaces

Research has shown that TMEM127 interacts with LAMTOR in an amino acid-dependent manner and decreases the LAMTOR1-vATPase association, while TMEM127-vATPase binding requires intact lysosomal acidification but is amino acid independent .