Recombinant Rat Transmembrane protein 174 (Tmem174)

What is Transmembrane Protein 174 (TMEM174) and what is its biological significance?

TMEM174, also known as immune response regulator, is a transmembrane protein that plays a crucial role in maintaining immune system balance by modulating immune cell activation and cytokine production. The protein is involved in various cell signaling pathways and has attracted research interest due to its potential involvement in cancer, autoimmune diseases, and inflammatory disorders . The full-length rat TMEM174 protein consists of 243 amino acids and contains multiple transmembrane domains .

Understanding TMEM174's function is essential for developing therapeutic strategies that manipulate immune responses in pathological conditions. Research methods typically involve using recombinant proteins and specific antibodies to investigate protein-protein interactions, signaling pathways, and cellular localization patterns.

What expression patterns does TMEM174 exhibit across different tissues?

TMEM174 demonstrates tissue-specific expression patterns with notable presence in renal tissues. RNA in situ hybridization studies have revealed differential expression across various renal tissue types, both normal and pathological. The protein exhibits particularly high expression levels in certain renal carcinoma subtypes, including squamous cell carcinoma with necrosis, papillary renal cell carcinoma, and transitional cell carcinoma .

In contrast, TMEM174 shows low expression rates in clear cell carcinoma, interstitial nephritis, and certain metastatic carcinomas. Expression is extremely weak in collecting duct carcinoma, Wilms' tumor, chronic pyelonephritis, acute pyelonephritis, cancer-adjacent normal renal tissue, and normal renal tissue . This differential expression pattern suggests tissue-specific regulation and potential involvement in pathological processes.

Methodologically, researchers can employ RNA in situ hybridization, quantitative PCR, and immunohistochemistry with specific antibodies to map expression patterns across various tissues and disease states.

What research tools are available for studying TMEM174?

Several research tools are available for investigating TMEM174:

• Antibodies: Polyclonal antibodies such as PACO30510 have been developed and validated for multiple applications including Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF). These antibodies typically show reactivity across human and mouse samples .

• Recombinant proteins: Full-length recombinant proteins are available, including Rat TMEM174 with N-terminal His-tags expressed in E. coli systems. These proteins can be used for generating antibodies, protein-protein interaction studies, and functional assays .

• Promoter constructs: Cloned promoter regions of various lengths have been developed for studying transcriptional regulation using reporter assays .

How is TMEM174 gene expression regulated at the transcriptional level?

TMEM174 transcriptional regulation involves complex interactions between multiple regulatory elements and transcription factors. Promoter analysis has identified several transcription factor binding sites within the promoter region, including CREB, AP-1, P300, NF-κB, and Oct1 .

Experimental evidence from electrophoretic mobility shift assays (EMSA) has demonstrated specific binding of CREB to the TMEM174 promoter. While AP-1 also binds to the promoter region, this binding appears to be non-specific, as it is only partially inhibited by competitive probes .

Dual luciferase reporter assays have revealed differential activity across various promoter fragments. Notably, fragments spanning -186 to +674 bp and -2,500 to +1 bp exhibit the highest levels of activity, suggesting that the core promoter region may be located within the -186 to +674 bp region. The regions spanning -186 to -466 bp and -700 to -890 bp appear to contain strong negative regulatory elements .

Researchers studying TMEM174 transcriptional regulation should consider:

• Designing reporter constructs with varying lengths of the promoter region

• Performing site-directed mutagenesis of predicted transcription factor binding sites

• Using EMSA and chromatin immunoprecipitation (ChIP) to confirm transcription factor binding

• Conducting cell-type specific analysis to account for differential regulation across tissues

What is the role of TMEM174 in renal cancer biology?

TMEM174 exhibits differential expression patterns across various renal cancer subtypes, suggesting potential involvement in carcinogenesis or tumor progression. RNA in situ hybridization studies have demonstrated high expression levels in squamous cell carcinoma with necrosis, papillary renal cell carcinoma, and transitional cell carcinoma .

In contrast, clear cell carcinoma, interstitial nephritis, undifferentiated carcinoma, and metastatic carcinomas show relatively lower expression levels. Collecting duct carcinoma, Wilms' tumor, and normal renal tissues exhibit extremely weak expression .

This differential expression pattern suggests that TMEM174 may play a significant role in the development and progression of specific renal carcinoma subtypes. The correlation between TMEM174 expression and cancer type might reflect its involvement in particular oncogenic pathways or cellular processes that contribute to tumor formation and growth.

Methodological approaches for investigating TMEM174's role in renal cancer include:

• Correlating expression levels with clinical outcomes and pathological features

• Knockdown or overexpression studies in renal cancer cell lines

• Analysis of downstream signaling pathways affected by TMEM174 modulation

• In vivo studies using xenograft models with modified TMEM174 expression

How does TMEM174 influence cell proliferation and signaling pathways?

TMEM174 has been identified as a potential regulator of cell proliferation through high-throughput cell screening technology. It activates AP-1 and promotes 293T cell proliferation . The protein appears to function as an immune response regulator by modulating immune cell activation and cytokine production, which contributes to maintaining immune system balance .

The mechanism by which TMEM174 promotes cell proliferation likely involves complex signaling pathways. Research has focused on both protein-level interactions and transcriptional regulation. At the transcriptional level, TMEM174 interacts with CREB and AP-1, transcription factors known to be involved in cell proliferation pathways .

To investigate TMEM174's influence on cell proliferation and signaling, researchers should consider:

• Analyzing phosphorylation events downstream of TMEM174 activation

• Performing gene expression profiling after TMEM174 modulation

• Conducting co-immunoprecipitation experiments to identify interacting proteins

• Using reporter assays to measure activation of relevant signaling pathways (e.g., AP-1, MAPK, NF-κB)

• Employing cell proliferation assays (e.g., MTT, BrdU incorporation) in response to TMEM174 overexpression or knockdown

What are the optimal conditions for expression and purification of recombinant Rat TMEM174?

Producing high-quality recombinant Rat TMEM174 requires careful optimization of expression and purification conditions. Based on established protocols, the following methodological approach is recommended:

• Expression system: E. coli has been successfully used for expressing full-length Rat TMEM174 (1-243aa) with an N-terminal His-tag . Consider using BL21(DE3) or Rosetta strains for improved expression of mammalian proteins.

• Expression conditions:

  • Induce at OD600 of 0.6-0.8 with 0.1-1.0 mM IPTG

  • Lower induction temperature (16-25°C) may improve protein folding

  • Extended expression time (overnight) at lower temperatures

• Lysis and purification:

  • Use Tris/PBS-based buffer systems at pH 8.0

  • Include protease inhibitors to prevent degradation

  • Employ affinity chromatography with Ni-NTA resin for His-tagged proteins

  • Consider detergent addition (mild non-ionic detergents like Triton X-100) for improved solubilization

• Storage and handling:

  • Store at -20°C/-80°C upon receipt

  • Aliquot to avoid repeated freeze-thaw cycles

  • Lyophilized powder should be reconstituted in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

Researchers should validate protein quality through SDS-PAGE analysis, with expected purity greater than 90% .

What experimental approaches can resolve contradictory findings in TMEM174 research?

When facing contradictory findings in TMEM174 research, several methodological approaches can help resolve discrepancies:

• Validate antibody specificity: Antibody cross-reactivity can lead to contradictory results. Validate antibodies using multiple techniques including Western blot, immunoprecipitation, and knockdown/knockout controls.

• Consider isoform-specific effects: The human TMEM174 gene produces at least two isoforms through alternative splicing . Differential isoform expression across tissues or experimental systems might explain contradictory findings.

• Evaluate cellular context: TMEM174 function may vary based on cell type or physiological state. Compare findings across multiple cell lines and primary cells, and consider the activation state of relevant pathways.

• Control for post-translational modifications: Assess how phosphorylation, glycosylation, or other modifications affect TMEM174 function and detection.

• Standardize recombinant protein preparation: Variations in protein folding, tag interference, or contaminants can affect functional studies. Compare proteins from different expression systems (bacterial, insect, mammalian) and with different tags.

• Comprehensive promoter analysis: When studying transcriptional regulation, analyze the entire promoter region rather than focusing on isolated segments, as both positive and negative regulatory elements influence gene expression .

• Statistical rigor: Ensure adequate statistical power, appropriate controls, and multiple biological replicates to validate findings.