Recombinant Mouse Transmembrane protein 170B (Tmem170b)

What is Transmembrane protein 170B (Tmem170b) and what is its molecular structure?

Tmem170b is a member of the TMEM170 family consisting of 132 amino acids with sequences that are highly conserved from invertebrates to mammals. It shares structural similarities with its important paralog, TMEM170A, which has been identified as a regulator of endoplasmic reticulum (ER) and nuclear envelope morphogenesis . Tmem170b is primarily localized in the plasma membrane and cytoplasmic regions, as demonstrated by immunohistochemistry (IHC) and immunofluorescence (IF) studies in pancreatic cancer cells .

The protein contains transmembrane domains that anchor it to cellular membranes, allowing it to participate in various cellular processes. For experimental studies, researchers should note that the full-length recombinant protein preserves the native conformation necessary for functional investigations.

How is Tmem170b expression regulated in normal versus pathological conditions?

Tmem170b shows differential expression patterns across various tissues and disease states:

• Cancer contexts: Tmem170b exhibits significantly lower expression in pancreatic adenocarcinoma (PAAD) tissues compared to non-tumorous tissues, as confirmed by multiple methodologies including RNA sequencing data from TCGA and GEO, immunohistochemistry, and RT-PCR . Similar downregulation has been observed in breast cancer, oral cancer, ovary cancer, and thyroid cancer .

• Inflammatory contexts: Interestingly, in sepsis, Tmem170b expression is significantly increased compared to controls, which contrasts with previous findings suggesting its downregulation in inflammatory responses after lipopolysaccharide (LPS) challenges .

This contextual expression pattern suggests tissue-specific and condition-specific regulatory mechanisms that warrant targeted investigation when designing expression studies.

What are the recommended methods for detecting and measuring Tmem170b expression?

Several validated methods are available for Tmem170b detection:

• RT-PCR: For mRNA quantification, the following primers have been validated:

  • Forward primer: 5′-TTCCTCTGGGCTCTCTTCTCT-3′

  • Reverse primer: 5′-CTGCTGCACTGGTAATCATCG-3′

  The relative expression levels should be calculated using the comparative CT (2^-ΔΔCT) method with β-Actin as a reference gene.

• Immunohistochemistry (IHC): Useful for tissue-level protein localization and expression analysis.

• Immunofluorescence (IF): Particularly valuable for subcellular localization studies, confirming Tmem170b's presence in plasma membrane and cytoplasmic regions .

• RNA sequencing: For genome-wide expression analysis, as demonstrated in studies using TCGA and GEO datasets .

For recombinant protein studies, Western blotting with specific antibodies against Tmem170b or epitope tags is recommended, especially when evaluating protein expression in transfection experiments.

How should researchers design experiments to investigate Tmem170b function in cancer models?

When designing experiments to investigate Tmem170b function in cancer models, researchers should consider:

• Cell line selection: Use paired normal and cancer cell lines from the same tissue origin. For pancreatic cancer research, studies have successfully employed pancreatic cancer cell lines alongside normal pancreatic duct epithelial cells as controls .

• Expression modulation: Implement both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches to comprehensively assess Tmem170b's role. This dual approach helps address directional effects and potential compensatory mechanisms.

• Functional assays: Include assays that measure:

  • Proliferation (e.g., MTT, EdU incorporation)

  • Migration/invasion (wound healing, transwell)

  • Colony formation

  • Apoptosis (Annexin V/PI staining)

  • Cell cycle analysis

• Xenograft models: For in vivo validation, consider both subcutaneous and orthotopic xenograft models using cells with modulated Tmem170b expression.

• Signaling pathway analysis: Investigate the effect on Wnt/β-catenin signaling, as Tmem170b has been shown to antagonize this protumorigenic pathway in breast cancer .

What approaches are available for generating recombinant mouse Tmem170b for research purposes?

Several approaches can be employed to generate recombinant mouse Tmem170b:

• Mammalian expression systems: These provide proper post-translational modifications and are recommended for functional studies. Common vectors include pcDNA3.1, pCMV, and lentiviral vectors for stable expression.

• Bacterial expression systems: While less optimal for full-length membrane proteins, they can be useful for producing specific domains for structural studies or antibody generation. Consider using fusion tags (His, GST) to facilitate purification.

• Cre/LoxP technology: For conditional gene manipulation in specific cell types or at specific time points. This approach requires generating transgenic mouse lines with floxed Tmem170b alleles .

• CRISPR/Cas9 gene editing: For creating knockout or knock-in mouse models or cell lines with modifications to the Tmem170b gene.

When selecting an expression system, consider the experimental objectives, required yield, and whether post-translational modifications are critical for the planned studies.

How can researchers optimize correlation and gene enrichment analysis for Tmem170b studies?

For comprehensive correlation and gene enrichment analysis related to Tmem170b function:

• Correlation analysis: Calculate Pearson correlation coefficients between Tmem170b and other mRNAs using datasets from TCGA or GEO. Select the top 300 genes most positively associated with Tmem170b for downstream enrichment analysis .

• Gene Ontology (GO) analysis: Utilize the functional annotation tool in the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov)[1].

• Pathway analysis: Implement Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) .

• Reactome Knowledgebase: Use this resource for both archiving biological processes and discovering functional relationships in data .

• R packages: Employ the "clusterProfiler" package with functions like gseGO, gseKEGG, and gsePathway for GSEA implementation .

This methodological framework enables comprehensive insights into the biological processes and signaling pathways associated with Tmem170b function.

How does Tmem170b influence the tumor immune microenvironment?

Tmem170b plays a significant role in modulating the tumor immune microenvironment:

• Immune cell infiltration: High Tmem170b expression correlates with an antitumor immune microenvironment characterized by increased infiltration of:

  • B cells

  • T cells

  • Dendritic cells

  • Monocytes

  • M1 macrophages

  • Neutrophils

  • Natural killer cells

• Suppressive immune populations: Conversely, high Tmem170b expression is associated with decreased infiltration of immunosuppressive populations:

  • Regulatory T cells (Tregs)

  • Myeloid-derived suppressor cells (MDSCs)

• Signaling pathways: Tmem170b is involved in immune-related gene sets and pathways, particularly:

  • Chemokine signaling pathways

  • Innate and adaptive immunity mechanisms

To investigate these relationships, researchers can utilize specialized tools like ESTIMATE (Estimation of Stromal Immune cells in MAlignant Tumor tissues using Expression data) and TIMER 2.0 (Tumor IMmune Estimation Resource) to analyze the correlation between Tmem170b expression and immune cell infiltration profiles .

What are the conflicting findings regarding Tmem170b in inflammatory conditions versus cancer?

Research has revealed contrasting roles for Tmem170b in different pathological contexts:

These divergent expression patterns suggest context-dependent regulation and function. Researchers should carefully consider disease context when designing studies and interpreting results. The mechanism behind these differential responses remains an important area for future investigation.

What is the significance of Tmem170b as a prognostic biomarker in cancer?

Multivariate analysis has identified Tmem170b as an independent prognostic indicator in pancreatic adenocarcinoma (PAAD):

This data demonstrates that low Tmem170b expression is significantly associated with poor prognosis in PAAD patients . Additionally:

These findings collectively suggest that Tmem170b could serve as a valuable prognostic biomarker across multiple cancer types, potentially guiding treatment decisions and risk stratification.

What are the common challenges in recombinant Tmem170b expression and purification?

Researchers working with recombinant Tmem170b may encounter several technical challenges:

• Protein solubility: As a transmembrane protein, Tmem170b contains hydrophobic domains that can reduce solubility during expression and purification. Consider using:

  • Detergents appropriate for membrane proteins (e.g., DDM, CHAPS)

  • Fusion partners that enhance solubility (e.g., MBP, SUMO)

  • Cell-free expression systems for difficult constructs

• Proper folding: Ensuring correct protein folding is critical for functional studies. Implementation of chaperone co-expression strategies can improve folding efficiency.

• Expression levels: Optimization of codon usage for the expression system and use of strong inducible promoters can help overcome low expression challenges.

• Protein degradation: Include protease inhibitors throughout the purification process and consider reducing the purification temperature to minimize degradation.

• Functional assays validation: Verify that the recombinant protein retains native activity through appropriate functional assays before proceeding with experimental applications.

How should researchers interpret conflicting data about Tmem170b function across different experimental systems?

When encountering conflicting data regarding Tmem170b function:

• Consider context dependency: The divergent expression patterns in cancer (downregulated) versus sepsis (upregulated) suggest that Tmem170b function may be highly context-dependent . Evaluate whether:

  • Different cell types or tissues were used

  • Different disease models or stages were examined

  • Different signaling pathways might be active in each context

• Methodological differences: Assess whether discrepancies might result from:

  • Different detection methods (protein vs. mRNA quantification)

  • Antibody specificity issues

  • Temporal differences in sampling or analysis

• Validation approaches: To resolve conflicts:

  • Use multiple, complementary techniques to verify findings

  • Include appropriate positive and negative controls

  • Perform dose-response or time-course experiments

  • Consider employing both in vitro and in vivo models

• Reporting standards: When publishing potentially conflicting findings, explicitly address previous contradictory results and propose potential mechanistic explanations for the differences.

What are the promising applications of Tmem170b as a therapeutic target?

Based on current understanding, several therapeutic applications warrant investigation:

• Cancer therapy: As a tumor suppressor gene, strategies to upregulate or restore Tmem170b function could potentially inhibit tumor growth and improve outcomes, particularly in:

  • Pancreatic adenocarcinoma

  • Breast carcinoma

  • Other cancers showing Tmem170b downregulation

• Immune modulation: Given Tmem170b's association with antitumor immune microenvironment and decreased immunosuppressive cell populations, it could serve as a target for immunotherapeutic approaches .

• Biomarker development: The prognostic value of Tmem170b expression suggests its utility as a biomarker for:

  • Patient stratification

  • Treatment response prediction

  • Disease monitoring

• Inflammatory disease management: The differential expression in sepsis points to potential applications in managing inflammatory conditions, though more research is needed to clarify its precise role .

• Combined therapeutic approaches: Consider combination strategies targeting both Tmem170b and related pathways, such as Wnt/β-catenin signaling, which Tmem170b has been shown to antagonize .

What genetic manipulation approaches are most suitable for in vivo Tmem170b studies?

For effective genetic manipulation of Tmem170b in vivo:

• Cre/LoxP technology: This system allows for spatial and temporal control of gene expression. While not specifically described for Tmem170b in the provided search results, the approach has been successfully applied to other transmembrane proteins like Tmem119 . For Tmem170b:

  • Consider generating floxed Tmem170b alleles

  • Select appropriate tissue-specific or inducible Cre lines

  • Validate recombination efficiency using reporter systems

• CRISPR/Cas9 genome editing: For generating:

  • Complete knockout models

  • Conditional knockout systems

  • Knock-in models with tagged versions or specific mutations

• Viral vector delivery: For spatially and temporally controlled expression:

  • Adeno-associated virus (AAV) for long-term expression

  • Lentivirus for integration and stable expression

  • Tissue-specific promoters for targeted expression

• Transgenic overexpression: To study gain-of-function effects by introducing exogenous Tmem170b under various promoters.

• RNA interference approaches: For transient knockdown using siRNA or more sustained effects with shRNA delivered via viral vectors.

Each approach has distinct advantages and limitations that should be considered in relation to the specific research questions being addressed.