TMEM79 Antibody

What is TMEM79 and what is its biological significance?

TMEM79 (transmembrane protein 79, also known as mattrin) is a protein with critical functions in maintaining skin barrier integrity. It has been genetically linked to atopic dermatitis (AD) in both mice and humans . TMEM79 is primarily expressed in keratinocytes but also found in sensory neurons, suggesting multi-tissue functionality . Its significance lies in its protective role against oxidative stress and inflammatory skin conditions. Loss-of-function mutations in TMEM79 lead to compromised skin barrier function, increased immune cell infiltration in the dermis, and enhanced scratching behavior characteristic of AD .

What is the molecular structure and cellular localization of TMEM79?

TMEM79 is a transmembrane protein that shows approximately 30% sequence similarity to membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEGs) such as MGST3 and MGST1 . This homology suggests potential functions in detoxifying reactive electrophiles. The protein contains specific functional residues, including R332 and Y339, which are critical for its protective effects against oxidative stress . Cellular localization studies using HA-tagged TMEM79 constructs have demonstrated its presence in both intracellular compartments and the cell membrane, with expression patterns varying between fixed and non-fixed cell preparations .

How does TMEM79 relate to skin barrier function and atopic dermatitis?

TMEM79 plays a crucial role in maintaining skin barrier integrity. Mutations in TMEM79 (such as in Tmem79 ma/ma mice) lead to dysregulated skin barrier function and spontaneous development of inflammatory conditions resembling atopic dermatitis . The protein's proposed mechanism involves protection against oxidative stress, as cells lacking functional TMEM79 show increased accumulation of reactive species when challenged with oxidants . Loss of TMEM79 function results in:

• Thickened dermis with immune cell infiltration

• Increased mast cell accumulation and degranulation

• Enhanced scratching behavior indicative of pruritus

• Elevated presence of IL-17-expressing γδ-T cells

These pathological changes collectively contribute to the development of AD-like symptoms.

What antibodies are available for TMEM79 detection and what are their applications?

For TMEM79 detection, researchers have successfully employed various antibody-based approaches. While the search results don't specify commercial antibodies, experiments have used antibodies against tagged versions of TMEM79 (such as HA-tagged constructs) . When selecting a TMEM79 antibody, researchers should consider:

• The species reactivity (mouse, rat, human) based on sequence conservation

• Detection method compatibility (Western blot, immunofluorescence, FACS)

• Epitope location (N-terminal vs. C-terminal) which may affect detection depending on protein orientation and potential truncated variants

Applications include immunohistochemistry to detect tissue expression patterns, Western blotting to confirm protein expression levels, and immunofluorescence microscopy to determine subcellular localization .

What protocols are recommended for immunohistochemical detection of TMEM79 in tissue samples?

Based on methodologies described in the research, optimal protocols for TMEM79 immunohistochemical detection include:

• Tissue fixation: Prepare tissue sections using either fresh-frozen or fixed (10% neutral buffered formalin) preparations

• Permeabilization: Use 0.1% Triton X-100 for intracellular epitope access

• Blocking: Apply 10% normal goat serum (NGS) for 1 hour at room temperature to reduce non-specific binding

• Primary antibody incubation: Dilute antibodies (typically 1:500) in PBS containing 0.1% Triton X-100 and 2.5% NGS, incubate for 1 hour at room temperature

• Washing: Perform three rinses with PBS

• Secondary antibody application: Use fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 594 at 1:1000 dilution) for 30 minutes

• Final washing: Three rinses with PBS

• Mounting: Apply mounting medium containing DAPI for nuclear counterstaining

For optimal imaging, confocal microscopy systems such as spinning-disk confocal setups provide superior resolution for evaluating TMEM79 localization patterns.

How can researchers validate TMEM79 antibody specificity?

Validating antibody specificity is critical for reliable TMEM79 research. Recommended validation approaches include:

• Genetic controls: Compare staining between wild-type and Tmem79-/- tissues or cells to confirm absence of signal in knockout samples

• Recombinant protein controls: Perform antibody testing with overexpressed tagged TMEM79 constructs where expression can be independently verified

• Epitope blocking: Pre-incubate antibody with immunizing peptide to confirm signal reduction

• Multiple antibody comparison: Use antibodies targeting different epitopes to confirm consistent localization patterns

• Western blot analysis: Verify single band of appropriate molecular weight (paired with knockout controls)

• RNA expression correlation: Compare protein detection patterns with mRNA expression data from RT-PCR or RNA-seq analyses

What mouse models are available for studying TMEM79 function?

Several genetically engineered mouse models have been developed to study TMEM79 function:

• Tmem79-/- (complete knockout): Generated through ES cell electroporation and homologous recombination, targeting vectors lacking exon 2 with the start codon. These mice exhibit AD-like phenotypes including thickened dermis, immune cell infiltration, and increased scratching behavior .

• Tmem79-KI conditional models: Created using Cre-lox technology with tissue-specific Cre drivers:

  • Tmem79-KI;K14-Cre: Keratinocyte-specific knockout

  • Tmem79-KI;Prph-Cre: Sensory neuron-specific knockout

  • Tmem79-KI;K14-Cre;Prph-Cre: Combined tissue knockout

• Tmem79 ma/ma mice: Carry a naturally occurring mutation in the Tmem79 gene, resulting in spontaneous skin and lung inflammation .

These models allow investigation of tissue-specific contributions to AD pathogenesis and can be used to test potential therapeutic interventions.

What cell culture systems are optimal for studying TMEM79 function?

Based on the research methodologies described, several cell culture systems have proven effective for TMEM79 studies:

• Primary keratinocytes: Isolated from wild-type and Tmem79-/- mice to study oxidative stress responses and cellular function

• HEK293T cells: Used for heterologous expression of wild-type and mutant TMEM79 constructs, particularly suitable for:

  • Subcellular localization studies using tagged constructs

  • Functional assays measuring protection against oxidative stress

  • Structure-function analyses through point mutations

• Sensory neuron cultures: Derived from dorsal root ganglia to examine TMEM79 expression in neuronal populations and responses to oxidative challenges

Culture conditions typically include DMEM supplemented with 10% FBS at 37°C with 5% CO₂. For transfection studies, Lipofectamine 3000 has been successfully employed with pcDNA3.1+ vectors containing TMEM79 coding sequences .

How should researchers approach TMEM79 gene expression analysis?

For comprehensive TMEM79 gene expression analysis, researchers should implement a multi-faceted approach:

• Primer design for PCR detection:

  • For wild-type Tmem79: Forward: 5′-AGC CTC CCA TTC CAA AGC-3′, Reverse: 5′-AGT CGT GCT GCT TCA TGT G-3′

  • For Tmem79-KI detection: Forward: 5′-CTG ATA TAC TGG TTG ACC TTT GCT-3′, Reverse: 5′-CCA GGC CTA CAA CTG TTC CA-3′

• RT-qPCR for expression quantification: Monitor relative expression changes under different conditions, such as oxidative stress challenges

• Reporter systems: The Tmem79-KI model incorporates eGFP fusion via a 2A peptide, enabling visualization of expression patterns in intact tissues

• Tissue collection considerations: Expression varies significantly between tissues, with prominent expression in epidermis and selected neuronal populations

• Expression regulation analysis: The Tmem79 gene contains a canonical antioxidant response element sequence that can be activated under oxidative stress conditions

How does TMEM79 modulate oxidative stress, and what experimental approaches can measure this function?

TMEM79 appears to protect cells from oxidative stress, possibly through a mechanism similar to membrane glutathione transferases. Experimental approaches to measure this function include:

• Cellular reactive species accumulation assay:

  • Load cells with cell-permeant 2,7-dichlorodihydrofluorescein diacetate (DCF/H₂DCFDA)

  • Expose cells to oxidants such as SIN-1, sodium nitroprusside, tert-butyl hydroperoxide, or H₂O₂

  • Monitor fluorescence (excitation 495nm/emission 529nm) over time (typically readings every 10 minutes for 1 hour)

  • Compare responses between wild-type TMEM79-expressing cells and controls or mutant variants

• Structure-function analysis:

  • Generate point mutations in putative active-site residues (R332S and Y339F) within the MAPEG-homologous region

  • Compare protective effects between wild-type and mutant proteins in oxidative challenge assays

• Antioxidant response measurement:

  • Challenge cells with oxidants and measure TMEM79 expression changes via RT-qPCR

  • Compare against known antioxidant response genes or genes lacking the antioxidant response element

  • Include positive controls such as N-acetyl cysteine to confirm assay specificity

These approaches collectively provide insights into TMEM79's role in cellular protection against oxidative damage.

What are the challenges in differentiating between direct and indirect effects of TMEM79 dysfunction?

Distinguishing direct from indirect effects of TMEM79 dysfunction presents several research challenges:

Researchers can address these challenges by combining multiple approaches, including timed inducible knockout systems, single-cell analyses to track response dynamics, and careful pharmacological intervention studies.

What is known about the interaction between TMEM79 and inflammatory pathways in atopic dermatitis?

The research reveals complex interactions between TMEM79 dysfunction and inflammatory pathways:

• Mast cell-mediated effects:
  TMEM79-deficient mice show increased mast cell presence and degranulation in the dermis . This leads to histaminergic itch through histamine receptor 1/histamine receptor 4 (H4R/H1R)-dependent mechanisms, potentially involving TRPV1-expressing afferents .

• T-cell involvement:
  Flow cytometry analyses revealed expansion of IL-17–expressing γδ-T cells in Tmem79-/- skin, contributing to the inflammatory phenotype .

• Prostaglandin signaling:
  Chronic treatment with cyclooxygenase inhibitors and an EP3 receptor antagonist reduced mast cell accumulation in Tmem79-/- mice, suggesting involvement of prostaglandin pathways in the inflammatory cascade .

• Inflammation control points:
  Therapeutic targeting of several pathway components showed efficacy in reducing AD symptoms:

  • PGE₂ synthesis/signaling inhibition

  • Histaminergic signaling blockade (H1R/H4R)

  • TRPA1 antagonism

These findings suggest that TMEM79 functions as an upstream regulator protecting against oxidative stress, which when compromised leads to activation of multiple inflammatory cascades contributing to AD pathogenesis.

What experimental design considerations are important when evaluating potential therapeutics targeting TMEM79-associated pathways?

When designing experiments to evaluate therapeutics for TMEM79-associated pathways, researchers should consider:

• Model selection:

  • Tmem79-/- complete knockout mice for systemic effects

  • Tissue-specific conditional knockouts to differentiate intervention points

  • In vitro systems for mechanism of action studies

  • Validation in human samples where feasible

• Intervention timing and duration:

  • Preventive (before symptom onset) versus therapeutic (after symptom establishment) testing

  • Acute versus chronic treatment regimens

  • Dose-response relationships

• Outcome measures:

  • Behavioral assessments (scratching frequency, duration)

  • Histological evaluations (skin thickness, immune cell infiltration)

  • Molecular markers (inflammatory cytokines, oxidative stress indicators)

  • Functional tests (skin barrier integrity, transepidermal water loss)

• Pathway targeting strategy:

  • Direct TMEM79 replacement or functional enhancement

  • Downstream intervention points:

    • Antioxidant supplementation

    • Anti-inflammatory agents

    • Anti-pruritic approaches (H1R/H4R antagonists)

    • PGE₂/EP3 signaling modulators

• Statistical considerations:

  • Power analyses based on effect sizes from preliminary data

  • Appropriate controls (vehicle, wild-type, positive control interventions)

  • Blinded assessment of outcomes where possible

How should researchers troubleshoot issues with TMEM79 antibody specificity and sensitivity?

When facing antibody-related challenges in TMEM79 research, systematic troubleshooting approaches include:

• Specificity verification:

  • Test antibodies on matched wild-type and Tmem79-/- samples

  • Perform peptide competition assays

  • Utilize epitope-tagged overexpression systems as positive controls

• Sensitivity optimization:

  • Evaluate multiple fixation protocols (10% NBF versus alternative fixatives)

  • Test different antigen retrieval methods for formalin-fixed tissues

  • Optimize antibody concentration through titration experiments

  • Explore signal amplification systems (tyramide signal amplification, polymer detection)

• Alternative detection strategies:

  • Use reporter systems (GFP fusion constructs) for live-cell imaging

  • Employ proximity ligation assays for protein interaction studies

  • Consider mass spectrometry-based approaches for unbiased protein detection

• Controls and validation:

  • Include isotype controls to assess non-specific binding

  • Verify patterns with multiple antibodies targeting different epitopes

  • Compare protein detection with mRNA expression data

What are the optimal methodologies for analyzing TMEM79 function in oxidative stress protection?

To effectively analyze TMEM79's role in oxidative stress protection, researchers should consider these methodological approaches:

• Reactive species detection:

  • Use cell-permeant 2,7-dichlorodihydrofluorescein diacetate (DCF) for general reactive species

  • Load cells with 5 μM DCF for 10 minutes at 37°C/5% CO₂

  • Wash cells twice with appropriate buffer (e.g., Ringer's solution)

  • Challenge with specific oxidants (SIN-1, sodium nitroprusside, tert-butyl hydroperoxide, H₂O₂)

  • Monitor fluorescence (excitation 495nm/emission 529nm) using plate readers at 10-minute intervals

• Experimental controls:

  • Include N-acetyl cysteine as a positive control antioxidant

  • Use oxidant concentration gradients to determine sensitivity thresholds

  • Compare wild-type TMEM79 to mutated versions (R332S, Y339F) lacking protective function

• Cell system considerations:

  • Primary cells from wild-type and knockout animals provide physiologically relevant contexts

  • Heterologous expression systems offer controlled protein level manipulation

  • Consider tissue-specific differences in antioxidant capacity

• Targeted pathway analysis:

  • Measure glutathione levels and oxidized/reduced glutathione ratios

  • Evaluate expression of antioxidant response genes alongside TMEM79

  • Assess mitochondrial function and ROS production

How can researchers accurately quantify and characterize TMEM79 expression across different tissue and cell types?

For comprehensive TMEM79 expression profiling across tissues and cells, researchers should implement:

• Multi-modal detection strategy:

  • mRNA quantification via RT-qPCR with validated primer sets

  • Protein detection using validated antibodies or reporter systems

  • Single-cell RNA sequencing for cell-type specific expression patterns

• Tissue processing optimization:

  • For epidermal samples: Separate epidermis from dermis using established protocols

  • For neuronal tissues: Consider rapid extraction and specialized fixation methods

  • For comparative studies: Ensure consistent processing across all samples

• Reporter mouse utilization:

  • The Tmem79-KI mouse model with GFP reporter enables direct visualization of expression

  • Flow cytometry of dissociated tissues allows quantitative assessment of expression levels

  • Tissue clearing techniques combined with light-sheet microscopy for 3D spatial mapping

• Expression regulation analysis:

  • Investigate antioxidant response element-mediated regulation under stress conditions

  • Measure relative expression changes following oxidant challenges

  • Compare TMEM79 expression kinetics with other stress-responsive genes

• Standardization approaches:

  • Use calibrated reference standards for quantitative PCR

  • Include multiple housekeeping genes for normalization

  • Apply consistent gating strategies for flow cytometry analyses