Recombinant Mouse Transmembrane protein 214 (Tmem214)

What is Mouse Transmembrane protein 214 (Tmem214) and what is its basic structure?

Tmem214 is a transmembrane protein encoded by the Tmem214 gene (Gene ID: 68796) in mice . The protein contains two putative transmembrane domains located at amino acids 480-500 and 616-636, with a large N-terminal domain that extends into the cytosol . The protein has a total length of approximately 689 amino acids, similar to its human ortholog . Structurally, Tmem214 is primarily localized to the outer membrane of the endoplasmic reticulum, where it serves as an anchor protein for other cellular components, particularly procaspase 4 .

How conserved is Tmem214 across different species?

Tmem214 is highly conserved across multiple species, indicating its fundamental biological importance. Orthologs have been identified in humans (TMEM214, Gene ID: 54867), rats (Tmem214, Gene ID: 362711), zebrafish (tmem214, Gene ID: 402840), and numerous other vertebrates including domestic guinea pig, naked mole-rat, chicken, dog, cow, domestic cat, domestic rabbit, and sheep . This high degree of conservation suggests that Tmem214 serves a critical evolutionary function in cellular processes across vertebrate species.

What are the main cellular functions of Tmem214?

Tmem214 primarily functions as a critical mediator of ER stress-induced apoptosis . The protein:

• Acts as an anchor for procaspase 4 (mouse homolog of human caspase 4) at the outer membrane of the ER

• Facilitates the activation of caspase-dependent apoptotic pathways in response to ER stress

• Functions independently of other ER stress response pathways such as CHOP induction and JNK phosphorylation

• Specifically mediates apoptosis induced by ER stressors but does not significantly affect apoptosis triggered by external factors or mitochondrion-dependent pathways

What experimental approaches are most effective for studying Tmem214 localization?

For investigating Tmem214 cellular localization, researchers should employ multiple complementary approaches:

• Immunofluorescent staining and confocal microscopy: This technique can demonstrate colocalization with ER markers (e.g., Sec61β) and lack of significant overlap with other organelle markers (Golgi, mitochondria) .

• Cell fractionation and immunoblot analysis: This approach can confirm that endogenous Tmem214 exists predominantly in the ER-containing membrane fraction, with minimal presence in mitochondria and cytosol .

• Trypsin-protection assays: Using purified membrane fractions, this technique can determine the membrane topology of Tmem214. In such assays, Tmem214 typically shows sensitivity to trypsin treatment (similar to procaspase 4), while ER lumen proteins like BiP remain protected, confirming Tmem214's localization to the outer membrane of the ER .

How can I effectively design knockdown experiments for Tmem214?

When designing Tmem214 knockdown experiments:

• RNAi design: Target specific regions of Tmem214 mRNA. Published research has successfully used multiple RNAi constructs that achieve significant reduction in Tmem214 expression .

• Validation of knockdown efficiency: Western blot analysis should be performed to confirm substantial reduction in Tmem214 protein levels. Effective knockdown has been demonstrated with at least 70-80% reduction in protein expression .

• Functional validation: Assess the impact of Tmem214 knockdown on ER stress-induced apoptosis using apoptosis-specific assays. Knockdown of Tmem214 should significantly inhibit apoptosis induced by ER stress inducers such as thapsigargin (TG) and brefeldin A (BFA) .

• Specificity controls: To demonstrate pathway specificity, include apoptosis inducers that operate through non-ER stress mechanisms (e.g., TNFα, actinomycin D, etoposide). Tmem214 knockdown should have minimal effect on these alternative apoptotic pathways .

How does Tmem214 interact with caspase pathways in ER stress-induced apoptosis?

Tmem214 serves as a crucial link between ER stress and caspase activation through the following mechanisms:

• Constitutive association: Tmem214 constitutively associates with procaspase 4 (mouse homolog of human caspase 4) at the outer membrane of the ER .

• Anchoring function: A specific fragment within the N-terminal cytoplasmic domain of Tmem214 (amino acids 176-354) is required for its interaction with procaspase 4, essentially anchoring this caspase to the ER membrane .

• Mutual dependency: Experimental evidence indicates that Tmem214 and caspase 4 are mutually dependent on each other in the ER stress-induced apoptotic pathway:

  • TMEM214-induced apoptosis is abolished by dominant negative mutants of procaspase 4

  • Caspase 4-induced apoptosis is inhibited by knockdown of TMEM214

  • Knockdown of TMEM214 inhibits the activation and cleavage of procaspase 4 by impairing its recruitment to the ER

• Specificity of interaction: The interaction is specific to procaspase 4, as Tmem214 knockdown does not significantly affect procaspase 8-induced apoptosis .

What methods can be used to study Tmem214-caspase interactions?

To investigate Tmem214-caspase interactions, consider these methodological approaches:

• Co-immunoprecipitation: This approach can confirm the physical interaction between Tmem214 and procaspase 4 in both overexpression systems and endogenous contexts .

• Domain mapping experiments: Using deletion constructs of Tmem214, researchers can identify specific regions required for procaspase 4 binding. Previous studies have pinpointed the N-terminal cytoplasmic domain (amino acids 176-354) as critical for this interaction .

• Mutational analysis: Creating dominant-negative mutants of procaspase 4 can help establish the functional significance of the interaction .

• Cellular fractionation with knockdown validation: This combined approach can demonstrate that Tmem214 is required for the association of procaspase 4 with the ER membrane .

What are the molecular mechanisms through which Tmem214 mediates ER stress-induced apoptosis?

The molecular mechanisms of Tmem214-mediated apoptosis involve:

How does Tmem214 expression correlate with sensitivity to ER stress in different cell types?

Research indicates a correlation between Tmem214 expression levels and cellular sensitivity to ER stress-induced apoptosis. Experimental observations across multiple cell lines (HeLa, HCT116, HepG2, and A549) suggest that the level of Tmem214 expression correlates with cellular sensitivity to thapsigargin (TG)-induced apoptosis . This correlation indicates that Tmem214 may be a determinant of cell type-specific responses to ER stress, potentially explaining differential sensitivity to ER stress-inducing conditions across tissues and cell types.

How can multi-omics approaches enhance our understanding of Tmem214 function?

Multi-omics approaches offer powerful strategies for comprehensive characterization of Tmem214:

• Transcriptomics: RNA-Seq analysis can identify changes in gene expression patterns following Tmem214 manipulation, revealing potential downstream effectors or compensatory mechanisms .

• Proteomics: Mass spectrometry-based approaches can:

  • Identify the complete interactome of Tmem214 beyond known interactions with procaspase 4

  • Characterize post-translational modifications that might regulate Tmem214 function

  • Quantify changes in protein abundance across cellular compartments

• Integrated analysis: Combining RNA-Seq, miRNA-Seq, and protein data (RPPA) can provide a systems-level understanding of how Tmem214 functions within broader cellular networks .

• Data integration tools: Specialized computational methods like DIABLO and NOLAS can be applied to integrate diverse omics datasets to identify key regulatory networks involving Tmem214 .

What is the potential significance of Tmem214 in disease models?

While specific disease associations for mouse Tmem214 require further investigation, its central role in ER stress-induced apoptosis suggests relevance to multiple pathological conditions:

• Neurodegenerative disorders: Given the importance of ER stress in conditions like Alzheimer's and Parkinson's diseases, Tmem214 may represent a potential therapeutic target .

• Cancer biology: The correlation between Tmem214 expression and sensitivity to ER stress-induced apoptosis suggests it may influence cancer cell survival under stress conditions. Multi-omics studies have begun exploring such connections in cancer datasets .

• Inflammatory conditions: As ER stress often occurs during inflammation, Tmem214's role in mediating apoptotic responses may be relevant to inflammatory pathologies .

• Metabolic disorders: Conditions characterized by elevated ER stress (e.g., diabetes, obesity) might be influenced by Tmem214-dependent apoptotic mechanisms .

What are the current challenges and future directions in Tmem214 research?

Current challenges and promising research directions include:

• Mechanistic gaps: While Tmem214 is known to mediate ER stress-induced apoptosis, the precise molecular events that activate this pathway during ER stress remain incompletely characterized .

• Tissue-specific functions: Further research is needed to elucidate potential tissue-specific roles of Tmem214 beyond the general mechanism of ER stress-induced apoptosis .

• Therapeutic potential: Investigating whether modulation of Tmem214 could offer therapeutic benefits in conditions characterized by dysregulated ER stress responses .

• Animal models: Development of Tmem214 knockout or conditional knockout mouse models would significantly advance understanding of its physiological functions in vivo.

• Cross-species variations: While the general function appears conserved, species-specific variations in Tmem214 regulation and interaction partners warrant further investigation .