Recombinant Mouse Transmembrane protein 209 (Tmem209)

What is Transmembrane Protein 209 (Tmem209)?

Tmem209 is an integral nuclear envelope protein that plays critical roles in cellular processes. In mouse models, it consists of 561 amino acids and functions in nuclear envelope organization, protein transport, and cellular signaling pathways. The protein is normally expressed at limited levels in healthy tissues, with notable expression in testis, but becomes significantly upregulated in various cancer tissues, suggesting its role in malignant transformation . The recombinant form often includes tags (such as His-tag) for purification and detection purposes .

What is the protein structure of mouse Tmem209?

Mouse Transmembrane protein 209 consists of 561 amino acids with a complex sequence containing multiple hydrophobic regions that facilitate its membrane integration. The full amino acid sequence is: MMQGDVSPNPSLIDRTIKMRKETETRKVVLAWGLLNVSMAGMIYTEMTGKLISTYYNVTYWPLWYIELALASLFSLNALFDFWRYFKYTVAPTSLVVSPGQQALLGLKQAVVQTTPPRDLAATQISPSPPSPSIQGQSVLSYSPSRSPSTSPKFATSCMTGYSPQLQGLSSGGLGSYSPGVTYSPVSGYNKLASFSLSPSSPYPTTVGPVESSGLRARYRSPPTVYNSPTDKEDYMTDLRTLDTFLRSEEEKQHRVKLGSPDSTSPSTSPTFWNYSRSVGDYAQTLKKFQYQLACRSQAPCANKDEADLISKQAAEEVWARVTMNRQLLDHMDSWTAKFRNWISETILVPLVQEIESVSTQMRRMGCPELQIGEASVTSLKQAALVRAPLIPTLNAIVQYLDLTPNQEYLFERIKELSQGGCMSSFRWNRGGDFKGRKWDTDLPTDSAIIMHVFCTYLDSRLPPHPKYPDGKTFTSQHFVQTPNKPDVTNENVFCIYQSAVNPPHYELIYQRHVYNLPKGRNNMFHTLLMFLYIIKTKESGMLGRVNLGLSGVNILWIFGE .

What are the known binding partners of Tmem209?

Research has identified several important binding partners of Tmem209. Most notably, mass spectrometric analysis revealed the nucleoporin protein NUP205 as a significant TMEM209-interacting protein in lung cancer cells . Additionally, in hepatocellular carcinoma cells, TMEM209 has been shown to bind to KPNB1 (Karyopherin Subunit Beta 1), thereby competitively blocking the interaction between KPNB1 and the E3 ubiquitin ligase RING finger and CHY zinc finger domain-containing protein 1 (RCHY1) . These protein-protein interactions appear critical to Tmem209's role in cancer progression.

How does Tmem209 contribute to cancer progression mechanisms?

Tmem209 promotes cancer progression through multiple mechanisms that vary by cancer type. In hepatocellular carcinoma (HCC), TMEM209 binds to KPNB1, blocking its interaction with the E3 ubiquitin ligase RCHY1. This prevents K48-associated ubiquitination degradation of KPNB1, ultimately activating the Wnt/β-catenin signaling pathway, which drives HCC progression . In lung cancer, TMEM209 interacts with NUP205, stabilizing this nucleoporin and increasing nuclear c-Myc levels, thus promoting cell proliferation . The overexpression of Tmem209 in various cancer types while being minimally expressed in normal tissues (except testis) suggests its potential role as a cancer-specific driver of malignant transformation.

What is the relationship between Tmem209 and the Wnt/β-catenin signaling pathway?

In hepatocellular carcinoma, TMEM209 has been shown to activate the Wnt/β-catenin signaling pathway through its interaction with KPNB1. By binding to KPNB1, TMEM209 competitively blocks the interaction between KPNB1 and the E3 ubiquitin ligase RCHY1, preventing the K48-associated ubiquitination degradation of KPNB1 . This stabilization of KPNB1 leads to the activation of the Wnt/β-catenin pathway, which is known to promote cell proliferation, invasion, and metastasis in various cancers. The relationship demonstrates how transmembrane proteins can influence critical oncogenic signaling pathways through protein-protein interactions rather than direct signaling.

How does the TMEM209-NUP205 interaction influence cellular processes?

The interaction between TMEM209 and the nucleoporin NUP205 has significant implications for cellular processes, particularly in cancer cells. Research has demonstrated that TMEM209 stabilizes NUP205 and increases the level of c-Myc in the nucleus . NUP205, as a component of the nuclear pore complex, is involved in nucleocytoplasmic transport. The stabilization of NUP205 by TMEM209 may alter nuclear transport dynamics, potentially facilitating the nuclear import of oncogenic factors or the export of tumor suppressors. The increased nuclear c-Myc levels, a known oncogenic transcription factor, drive cell proliferation, metabolic reprogramming, and other hallmarks of cancer.

What is the functional significance of TMEM209 overexpression in different cancer types?

TMEM209 overexpression has been documented in multiple cancer types, with varying functional implications. In lung cancer, it drives cellular proliferation and survival, with knockdown experiments resulting in growth inhibition and G1 cell cycle arrest . In hepatocellular carcinoma, TMEM209 overexpression promotes proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and facilitates tumor growth and metastasis in xenograft models . The consistent finding across cancer types is that TMEM209 overexpression correlates with poor prognosis and aggressive disease characteristics. The mechanism appears to involve both nuclear envelope functions and cytoplasmic signaling pathway modulation, suggesting that TMEM209 acts as a multifunctional oncogenic driver.

What are the optimal conditions for expressing recombinant mouse Tmem209 in bacterial systems?

For the successful expression of recombinant mouse Tmem209 in bacterial systems, E. coli has been demonstrated as an effective host . When expressing the full-length protein (1-561 amino acids), it is recommended to use an N-terminal His-tag for purification purposes. The expression construct should contain the complete coding sequence with appropriate bacterial promoters. After expression, the protein is typically obtained in lyophilized powder form and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, the addition of 5-50% glycerol (with 50% being the standard concentration) is recommended, followed by aliquoting and storage at -20°C/-80°C to prevent repeated freeze-thaw cycles that could compromise protein integrity.

What methods are most effective for studying Tmem209 protein-protein interactions?

Several methodological approaches have proven effective for investigating Tmem209 protein-protein interactions:

• Mass Spectrometry: This technique has successfully identified NUP205 as a TMEM209-interacting protein in lung cancer research .

• Co-immunoprecipitation (Co-IP): This method can verify direct binding between Tmem209 and potential interacting partners such as KPNB1 and NUP205.

• Proximity Ligation Assays (PLA): These provide spatial resolution of protein interactions within cells, helping to determine where in the cell Tmem209 interacts with its binding partners.

• Yeast Two-Hybrid Systems: While not explicitly mentioned in the search results, this approach can screen for novel Tmem209 interacting proteins.

• Pull-down Assays: Using recombinant His-tagged Tmem209 , researchers can perform pull-down experiments to identify and validate binding partners.

These methods have successfully elucidated the interaction networks of Tmem209, including its binding to KPNB1 in hepatocellular carcinoma and NUP205 in lung cancer .

How can RNA interference be effectively designed to study Tmem209 function?

RNA interference (RNAi) has been successfully employed to investigate Tmem209 function in cancer cells. When designing siRNAs against Tmem209, researchers should:

• Design multiple siRNAs: In lung cancer studies, researchers used at least two different siRNAs (si-TMEM209-#1 and si-TMEM209-#2) to ensure result consistency and minimize off-target effects .

• Include appropriate controls: Both negative controls (si-EGFP or LUC) and experimental controls (such as testing siRNAs in cells with low Tmem209 expression) should be used to validate specificity .

• Validate knockdown efficiency: Western blotting should be performed to confirm significant reduction in Tmem209 protein levels after siRNA transfection .

• Assess functional endpoints: Following Tmem209 knockdown, researchers should evaluate multiple functional outcomes, including colony formation assays, viability (MTT) assays, and flow cytometric analysis for cell cycle effects .

This approach has successfully demonstrated that Tmem209 knockdown results in growth inhibition and G1 arrest in lung cancer cells, confirming its critical role in cancer cell proliferation.

What techniques are most suitable for analyzing Tmem209's role in tumor progression in vivo?

To analyze Tmem209's role in tumor progression in vivo, several experimental approaches have proven effective:

• Xenograft Models: Implanting cancer cells with modified Tmem209 expression (overexpression or knockdown) into immunocompromised mice has successfully demonstrated Tmem209's role in tumor growth and metastasis, as shown in hepatocellular carcinoma studies .

• Genetically Engineered Mouse Models: Though not explicitly mentioned in the search results, conditional knockout or overexpression of Tmem209 in specific tissues could provide insights into its role in tumor initiation and progression.

• Immunohistochemistry: This technique can be used to analyze Tmem209 expression patterns in tumor tissues and correlate with clinical outcomes.

• In vivo Imaging: Techniques such as bioluminescence imaging can track tumor growth and metastasis in real-time in animals with Tmem209-modified cancer cells.

• Molecular Analysis of Tumor Tissues: RNA-seq, proteomics, and pathway analysis of tumors with altered Tmem209 expression can reveal downstream molecular changes.

These approaches have successfully established Tmem209's role in facilitating tumor growth and metastasis in xenograft models of hepatocellular carcinoma .

How can researchers differentiate between the cellular effects of Tmem209 and other similar transmembrane proteins?

Differentiating between the cellular effects of Tmem209 and other transmembrane proteins requires a multi-faceted experimental approach:

• Specificity Controls: When studying Tmem209 knockdown effects, researchers should verify that siRNAs do not affect the expression of other transmembrane proteins. For example, in lung cancer research, siRNAs against TMEM209 did not affect cell viability in SBC-3 cells with minimal endogenous TMEM209 expression, confirming specificity .

• Rescue Experiments: After knockdown of Tmem209, reintroduction of siRNA-resistant Tmem209 should restore the phenotype if effects are specific.

• Domain-Specific Analysis: Creating truncated or mutated versions of Tmem209 can help identify which protein domains are responsible for specific cellular effects.

• Comparative Studies: Directly comparing the effects of Tmem209 manipulation with that of other transmembrane proteins (like TMEM9 ) in the same experimental system can highlight unique functions.

• Binding Partner Specificity: Identifying unique binding partners, such as NUP205 for TMEM209 in lung cancer or KPNB1 in hepatocellular carcinoma , helps distinguish its specific signaling pathways.

These approaches collectively enable researchers to attribute cellular effects specifically to Tmem209 rather than to general perturbations of membrane proteins.

What are the key considerations when interpreting Tmem209 expression data across different tumor types?

When interpreting Tmem209 expression data across different tumor types, researchers should consider several factors:

• Tissue-Specific Baseline Expression: Normal Tmem209 expression is limited primarily to testis, making its upregulation in tumors potentially significant . Researchers should always compare tumor expression to the appropriate normal tissue.

• Subcellular Localization Differences: Tmem209 localizes to the nuclear envelope, Golgi apparatus, and cytoplasm in lung cancer cells , but localization may vary in other cancer types, potentially impacting function.

• Interaction with Tissue-Specific Pathways: Tmem209 interacts with KPNB1 in hepatocellular carcinoma to activate Wnt/β-catenin signaling , while in lung cancer, it interacts with NUP205 . These tissue-specific interaction networks should be considered.

• Correlation with Clinical Outcomes: Expression levels should be correlated with patient survival, tumor stage, and other clinical parameters to understand prognostic significance.

• Multi-Omics Integration: Combining Tmem209 expression data with mutation, methylation, and proteomic data provides a more comprehensive understanding of its role in different cancer types.

These considerations help researchers properly contextualize Tmem209 expression data and avoid overgeneralizing findings from one cancer type to another.

How should researchers address contradictory findings regarding Tmem209 function in different experimental systems?

When addressing contradictory findings regarding Tmem209 function across different experimental systems, researchers should:

• Examine Experimental Conditions: Different cell lines, culture conditions, or in vivo models may influence Tmem209 function. For example, the effects of TMEM209 knockdown were validated in multiple lung cancer cell lines (LC319 and SBC-5) but not in SBC-3 cells with low endogenous TMEM209 expression .

• Consider Cell Type-Specific Context: Tmem209 may have different binding partners and downstream effects depending on the cellular context. The TMEM209-KPNB1 interaction in hepatocellular carcinoma differs from the TMEM209-NUP205 interaction in lung cancer .

• Evaluate Technical Approaches: Different knockdown efficiencies, overexpression levels, or analytical methods may contribute to discrepancies.

• Assess Protein Modifications: Post-translational modifications might affect Tmem209 function differently across experimental systems.

• Design Bridging Experiments: When contradictions arise, design experiments specifically to address and resolve discrepancies, such as using the same methodological approach across different cell types.

By systematically addressing these factors, researchers can reconcile contradictory findings and develop a more nuanced understanding of Tmem209's context-dependent functions.

What statistical approaches are most appropriate for analyzing the correlation between Tmem209 expression and clinical outcomes?

When analyzing correlations between Tmem209 expression and clinical outcomes, several statistical approaches are recommended:

• Kaplan-Meier Survival Analysis: This method can effectively demonstrate the relationship between Tmem209 expression levels and patient survival. Studies have shown that TMEM209 upregulation correlates with reduced survival duration in hepatocellular carcinoma patients .

• Cox Proportional Hazards Regression: This multivariate analysis can determine if Tmem209 expression is an independent prognostic factor while accounting for other clinical variables.

• ROC Curve Analysis: This approach can assess the sensitivity and specificity of Tmem209 as a biomarker for predicting clinical outcomes.

• Correlation Analyses: Spearman or Pearson correlation can evaluate associations between Tmem209 expression and continuous clinical variables or other molecular markers.

• Machine Learning Approaches: More advanced computational methods can integrate Tmem209 expression with other molecular data to build predictive models for patient outcomes.

These statistical methods have been successfully applied in cancer studies, revealing that TMEM209 upregulation correlates with poorer prognosis in hepatocellular carcinoma and potentially in other cancer types.

What are the recommended storage and handling conditions for recombinant mouse Tmem209?

For optimal stability and activity of recombinant mouse Tmem209, the following storage and handling conditions are recommended:

• Storage Temperature: Store at -20°C/-80°C upon receipt .

• Aliquoting: Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles .

• Reconstitution: Before use, briefly centrifuge the vial to bring contents to the bottom. Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage Buffer: For reconstituted protein, add glycerol to a final concentration of 5-50% (with 50% being standard) and aliquot for long-term storage .

• Working Storage: For short-term use, working aliquots can be stored at 4°C for up to one week .

• Freeze-Thaw Cycles: Repeated freezing and thawing should be avoided as it may lead to protein denaturation and loss of activity .

These recommendations ensure the maintenance of protein integrity and biological activity for experimental purposes.

What analytical techniques are most suitable for verifying the purity and integrity of recombinant Tmem209?

To verify the purity and integrity of recombinant mouse Tmem209, several analytical techniques are recommended:

• SDS-PAGE: This is the primary method for assessing protein purity, with recombinant Tmem209 typically showing purity greater than 90% .

• Western Blotting: Using anti-His antibodies (for His-tagged Tmem209) or specific anti-Tmem209 antibodies can confirm protein identity and integrity.

• Mass Spectrometry: This technique can verify the molecular weight and sequence of the recombinant protein, particularly useful for confirming full-length expression (1-561 amino acids).

• Size Exclusion Chromatography: This method can assess protein aggregation state and homogeneity.

• Circular Dichroism: While not explicitly mentioned in the search results, this technique can evaluate the secondary structure of the protein to ensure proper folding.

• Functional Assays: Binding assays with known interaction partners like NUP205 or KPNB1 can confirm that the recombinant protein retains its functional properties.

These analytical approaches collectively ensure that the recombinant Tmem209 protein meets the quality standards required for reliable experimental results.