Recombinant Rat Transmembrane protein 214 (Tmem214)

What is Transmembrane Protein 214 and what are its structural characteristics?

Transmembrane protein 214 (Tmem214) is a 77kDa membrane protein that is widely expressed at high levels across various tissues. In rats, the gene encoding this protein maps to chromosome 2. The protein contains two transmembrane domains located at the C-terminus (specifically at amino acids 480-500 and 616-636 in the human ortholog), with a large N-terminal domain that extends into the cytosol . Structure-function analyses have determined that the N-terminal cytoplasmic domain (specifically amino acids 176-354 in humans) is crucial for its interaction with other proteins, particularly procaspase 4. Interestingly, deletion studies have shown that while removal of either transmembrane domain doesn't impair ER localization or apoptotic function, deletion of either the N-terminal binding region or both C-terminal transmembrane domains abolishes its ability to induce apoptosis .

Where is Tmem214 primarily localized in cells?

Tmem214 is predominantly localized to the endoplasmic reticulum (ER), specifically on its outer membrane. This localization has been confirmed through multiple experimental approaches including immunofluorescent staining, cell fractionation analysis, and trypsin-protection assays. Confocal microscopy analysis has demonstrated that Tmem214 colocalizes primarily with the ER marker Sec61β, with minimal overlap with Golgi body or mitochondrial markers . Cell fractionation studies further support this localization pattern, showing that endogenous Tmem214 exists mostly in the ER-containing membrane fraction, with only low levels detected in mitochondria and none in the cytosol . This specific localization is critical for its function in ER stress-induced apoptotic pathways.

What are the known molecular interactions of Tmem214?

Tmem214 has been identified to interact with several proteins that contribute to its cellular functions. Most notably, Tmem214 constitutively associates with procaspase 4, serving as an anchor that recruits this protease to the ER membrane . This interaction occurs through the N-terminal cytoplasmic domain (amino acids 176-354) of Tmem214 and is essential for its role in mediating ER stress-induced apoptosis. Additionally, Tmem214 interacts with LSM1, an SM-like protein, forming a stable heteromer present in tri-snRNP particles that are important for pre-mRNA splicing . These molecular interactions provide insight into the dual functionality of Tmem214 in both apoptotic signaling and RNA processing, though the relationship between these functions remains to be fully elucidated through targeted interaction studies.

How can researchers validate the expression of Tmem214 in experimental systems?

For validating Tmem214 expression, researchers should employ a multi-modal approach combining protein and transcript analysis. Western blot analysis using validated antibodies such as the polyclonal antibody PA5-20792 (which reacts with human, mouse, and rat Tmem214) can be used for protein detection . When performing Western blots, rat brain tissue lysate serves as an effective positive control . RT-qPCR can be used to quantify mRNA expression levels, with primers targeting conserved regions of the transcript. For immunolocalization studies, researchers should perform co-staining with established ER markers such as Sec61β to confirm proper localization . When using recombinant systems, epitope tags such as FLAG can be incorporated to facilitate detection and purification of the expressed protein. Validation should always include appropriate controls and, when possible, multiple detection methods to ensure specificity.

What is the role of Tmem214 in ER stress-induced apoptosis?

Tmem214 functions as a critical mediator in endoplasmic reticulum (ER) stress-induced apoptosis through a mechanism independent of the CHOP and JNK pathways. Experimental evidence demonstrates that overexpression of Tmem214 induces apoptosis characterized by cellular morphological changes, positive annexin V staining, and DNA fragmentation . Conversely, knockdown of Tmem214 significantly inhibits apoptosis triggered by ER stress inducers such as thapsigargin (TG) and brefeldin A (BFA), while showing no effect on apoptosis induced by external factors like TNFα or DNA damage agents such as actinomycin D and etoposide .

The mechanism involves Tmem214 serving as an anchor protein that constitutively associates with procaspase 4 at the ER outer membrane. During ER stress, this positioning facilitates the activation and cleavage of procaspase 4, initiating the apoptotic cascade. Importantly, the apoptotic function of Tmem214 depends on this interaction, as evidenced by the fact that Tmem214-induced apoptosis is abolished by a dominant negative mutant of procaspase 4, while caspase 4-induced apoptosis is inhibited by knockdown of Tmem214 .

It's worth noting that Tmem214-mediated apoptosis represents only one pathway in ER stress-induced cell death, explaining why knockdown of Tmem214 does not completely abolish ER stress-induced apoptosis. Experimental data confirms that Tmem214 operates independently of other established ER stress-induced apoptotic mechanisms involving CHOP induction and JNK phosphorylation .

What methodologies are most effective for studying Tmem214 function in ER stress responses?

Investigating Tmem214 function in ER stress responses requires a multifaceted methodological approach:

• Gene Modulation Techniques:

  • RNA interference (RNAi) using validated constructs targeting TMEM214 (similar to the #2 and #3 RNAi plasmids described in the literature)

  • CRISPR-Cas9 genome editing for complete knockout studies

  • Overexpression systems using expression vectors with appropriate promoters

• ER Stress Induction:

  • Pharmacological inducers: thapsigargin (TG) and brefeldin A (BFA) at optimized concentrations

  • Compare with other apoptotic stimuli (TNFα, actinomycin D, etoposide) as controls to confirm specificity

• Apoptosis Assessment:

  • Morphological analysis (round-up morphology, detachment from culture dishes)

  • Annexin V staining coupled with flow cytometry quantification

  • DNA fragmentation assays

  • PARP cleavage detection by Western blot

• Protein Interaction Studies:

  • Co-immunoprecipitation with procaspase 4 antibodies

  • Proximity ligation assays to visualize endogenous protein-protein interactions in situ

  • Domain mapping using truncated mutants to identify critical interaction regions

• Subcellular Localization:

  • Immunofluorescence microscopy with co-staining for ER markers (e.g., Sec61β)

  • Cell fractionation followed by Western blot analysis

  • Trypsin-protection assays to determine membrane topology

• Functional Readouts:

  • Caspase 4 activation assays

  • Use of specific caspase inhibitors (e.g., Z-LEVD for caspase 4)

  • Analysis of downstream apoptotic events

These methodologies should be applied in complementary fashion, with appropriate controls, to comprehensively characterize Tmem214 function in ER stress responses.

How does rat Tmem214 compare structurally and functionally to its human ortholog?

Rat Tmem214 shares significant structural homology with its human ortholog, though with some species-specific differences that may affect experimental design and interpretation. The rat Tmem214 protein is identified by the UniProt ID A1L1L2, while the human ortholog is Q6NUQ4 . Both proteins maintain the characteristic dual transmembrane domains at the C-terminus and a large N-terminal cytoplasmic region that mediates protein interactions.

When designing experiments using recombinant rat Tmem214, researchers should consider these species-specific differences, especially when translating findings between rodent models and human systems. Cross-reactivity of antibodies between species should be verified; for instance, the polyclonal antibody PA5-20792 has been validated to recognize human, mouse, and rat Tmem214 , making it suitable for comparative studies.

What are the implications of chemical modulation of Tmem214 expression?

Chemical compounds can significantly alter Tmem214 expression, providing valuable experimental tools but also introducing potential confounding factors in research. Most notably, 1,2-dimethylhydrazine has been documented to increase Tmem214 expression in rat models . This chemical modulation has important implications for both experimental design and interpretation of results.

In toxicological and cancer research contexts, the relationship between 1,2-dimethylhydrazine (a known carcinogen) and Tmem214 upregulation suggests a potential link between aberrant Tmem214 expression and carcinogenesis pathways . This connection warrants further investigation, particularly regarding whether Tmem214 upregulation represents a protective cellular response or contributes to pathological processes.

Methodologically, researchers studying Tmem214 should consider screening experimental compounds for unintended effects on Tmem214 expression, particularly in studies where ER stress and apoptosis are relevant endpoints. Similarly, when interpreting phenotypes resulting from chemical treatments known to affect Tmem214 levels, the contribution of altered Tmem214 expression to the observed outcomes should be carefully evaluated.

What techniques are most effective for producing and purifying recombinant rat Tmem214 for research applications?

Production and purification of recombinant rat Tmem214 presents significant challenges due to its transmembrane nature, but several approaches can yield functional protein for research applications:

• Expression System Selection:

  • Mammalian expression systems (HEK293, CHO cells) generally provide proper folding and post-translational modifications

  • Baculovirus-insect cell systems offer a compromise between proper folding and yield

  • Bacterial systems may be suitable for expression of soluble domains (e.g., the N-terminal region)

• Construct Design Considerations:

  • Incorporate affinity tags (His6, FLAG, GST) for purification, preferably at the N-terminus to avoid interfering with transmembrane domains

  • Consider fusion proteins with MBP or SUMO to enhance solubility

  • For structural studies, design constructs of the soluble N-terminal domain (amino acids 1-110) as used for antibody production

• Detergent-Based Extraction Methods:

  • Use mild non-ionic detergents (DDM, LMNG) for initial solubilization

  • Consider testing multiple detergent conditions in parallel

  • Implement a detergent screening approach to identify optimal solubilization conditions

• Purification Strategy:

  • Two-step affinity chromatography (e.g., Ni-NTA followed by anti-FLAG)

  • Size exclusion chromatography to remove aggregates

  • For structural studies, consider amphipol exchange or nanodisc reconstitution

• Functional Validation:

  • Verify proper folding through circular dichroism

  • Confirm interaction with known binding partners (e.g., procaspase 4) through pull-down assays

  • Assess ability to induce apoptosis when reintroduced into cells

• Storage Considerations:

  • Include stabilizing agents (glycerol, specific lipids)

  • Aliquot and flash-freeze to minimize freeze-thaw cycles

  • Validate activity after storage

When utilizing recombinant rat Tmem214, researchers should confirm that the protein maintains its native properties, particularly its ability to localize to the ER membrane and interact with relevant binding partners like procaspase 4 or LSM1.

How can Tmem214 research contribute to understanding disease mechanisms?

Tmem214 research has significant implications for understanding various disease mechanisms, particularly those involving ER stress and apoptotic dysregulation. The critical role of Tmem214 in ER stress-induced apoptosis positions it as a potential mediator in conditions where this pathway is implicated, including neurodegenerative diseases, diabetes, and certain cancers .

Neurodegenerative disorders such as Alzheimer's and Parkinson's diseases frequently involve ER stress and subsequent neuronal apoptosis. Understanding how Tmem214 regulates this process could provide insights into disease progression and potential therapeutic targets. The high expression of Tmem214 in brain tissue further supports its relevance to neurological conditions .

In cancer research, the dual nature of ER stress (protective versus pro-apoptotic) makes Tmem214 an interesting subject of investigation. Its increased expression following exposure to 1,2-dimethylhydrazine, a known carcinogen, suggests potential involvement in carcinogenesis processes . Additionally, researchers have noted that Tmem214 may be involved in host responses to infectious diseases, including viral infections like Dengue fever, West Nile fever, and yellow fever .

Methodologically, disease-relevant research on Tmem214 should incorporate:

• Patient-derived samples to assess expression levels in diseased versus healthy tissues

• Disease-specific cellular and animal models with modulated Tmem214 expression

• Pharmacological modulators of the ER stress response to determine therapeutic potential

• Multi-omics approaches to position Tmem214 within broader disease networks

What are the current challenges and knowledge gaps in Tmem214 research?

Despite significant progress, several challenges and knowledge gaps persist in Tmem214 research:

• Regulatory Mechanisms:
  The mechanisms regulating Tmem214 expression and activation during ER stress remain poorly understood. While research indicates that protein levels do not markedly change during ER stress, other regulatory mechanisms such as conformational changes, oligomerization, or post-translational modifications may play crucial roles . Methodological approaches to address this gap would include comprehensive post-translational modification analysis, structural studies, and real-time monitoring of Tmem214 dynamics during ER stress.

• Functional Duality:
  Tmem214 has been implicated in both mRNA processing through its interaction with LSM1 and in apoptotic pathways through procaspase 4 binding . How these seemingly distinct functions are coordinated or whether they represent cell type-specific roles remains unclear. Single-cell analyses examining co-expression patterns and conditional knockout models could help resolve this duality.

• Species-Specific Differences:
  While the human and rat orthologs of Tmem214 share structural similarities, functional differences may exist, particularly regarding interaction partners (human procaspase 4 versus rodent caspase-12). Comparative studies using both human and rat systems are needed to accurately translate findings between models.

• Therapeutic Potential:
  Whether Tmem214 represents a viable therapeutic target for conditions involving dysregulated ER stress responses remains unexplored. High-throughput screening for small molecule modulators of Tmem214 function, coupled with validation in disease models, would address this gap.

• Structural Characterization:
  Detailed structural information, particularly regarding the transmembrane domains and their organization within the ER membrane, is lacking. Cryo-EM or crystallography studies of the full-length protein or its domains would significantly advance understanding of its function.

Addressing these challenges will require multidisciplinary approaches combining advanced structural biology, systems biology, and translational research methodologies.

What are the optimal conditions for studying Tmem214-mediated apoptosis in cell culture models?

To effectively study Tmem214-mediated apoptosis in cell culture models, researchers should consider the following optimized experimental conditions:

• Cell Line Selection:

  • HeLa cells have been successfully used in Tmem214 knockdown studies and show clear apoptotic responses to ER stressors

  • Other validated cell lines include HCT116, HepG2, and A549, which demonstrate correlation between Tmem214 levels and sensitivity to ER stress inducers like thapsigargin (TG)

  • Primary rat cells (particularly neurons) may provide physiologically relevant contexts given Tmem214's high expression in brain tissue

• ER Stress Induction Protocols:

  • Thapsigargin (TG): 1-5 μM for 24-48 hours

  • Brefeldin A (BFA): 1-10 μg/ml for 24-48 hours

  • Include non-ER stress apoptotic inducers as controls (TNFα, actinomycin D, etoposide) to confirm specificity

• Tmem214 Modulation Approaches:

  • For knockdown: RNAi plasmids targeting different sites of Tmem214 mRNA, delivered via retroviral transduction for stable expression

  • For overexpression: Expression vectors with CMV or similar strong promoters, with careful titration to prevent excessive toxicity

• Apoptosis Detection Methods:

  • Annexin V/PI staining coupled with flow cytometry (provides quantitative assessment)

  • TUNEL assay for DNA fragmentation

  • Caspase activity assays specifically measuring caspase 4 activity

  • Western blot for cleaved PARP as an apoptotic marker

• Controls and Validation:

  • Include vector-only controls for overexpression studies

  • Use non-targeting shRNA controls for knockdown experiments

  • Validate Tmem214 modulation by Western blot and/or qRT-PCR

  • For mechanistic studies, include caspase 4 inhibitor Z-LEVD to confirm pathway specificity

• Timeline Considerations:

  • Monitor cellular responses at multiple time points (6, 12, 24, 48 hours)

  • For overexpression studies, begin monitoring no later than 20 hours post-transfection, when morphological changes become apparent

These optimized conditions should enable researchers to reproducibly study Tmem214-mediated apoptosis while controlling for non-specific effects and ensuring pathway specificity.

What antibodies and detection methods are most reliable for Tmem214 research?

For reliable detection of Tmem214 in research applications, the following antibodies and detection methods have been validated:

Validated Antibodies:

• Polyclonal Antibodies:

  • Invitrogen Anti-TMEM214 Polyclonal (PA5-20792) has been confirmed to react with human, mouse, and rat Tmem214 in Western blot applications

  • Custom rabbit anti-TMEM214 antiserum raised against recombinant human TMEM214 (amino acids 1-110) has been used successfully in immunoprecipitation and Western blot applications

• Tagged Protein Detection:

  • Anti-FLAG antibodies (Sigma) for detection of FLAG-tagged recombinant Tmem214

  • Anti-cherry antibodies for detection of cherry-tagged Tmem214 in localization studies

Optimal Detection Methods:

• Western Blot Protocol:

  • Sample preparation: Lyse cells in RIPA buffer with protease inhibitors

  • Protein loading: 20-50 μg total protein per lane

  • Positive control: Rat brain tissue lysate is recommended

  • Blocking: 5% non-fat milk in TBST for 1 hour

  • Primary antibody: Anti-TMEM214 (PA5-20792) at 1:1000 dilution, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000

  • Expected size: 77 kDa main band

• Immunofluorescence Protocol:

  • Fixation: 4% paraformaldehyde for 15 minutes

  • Permeabilization: 0.1% Triton X-100 for 10 minutes

  • Blocking: 3% BSA in PBS for 1 hour

  • Co-staining: Include ER marker (e.g., Sec61β) to confirm localization

  • Secondary antibodies: Alexa Fluor-conjugated antibodies for dual labeling

  • Counterstain: DAPI for nuclear visualization

• Co-immunoprecipitation:

  • Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA with protease inhibitors

  • Pre-clearing: Protein A/G beads for 1 hour

  • Immunoprecipitation: Anti-TMEM214 antibody or anti-tag antibody overnight at 4°C

  • Detection of binding partners: Anti-procaspase 4 antibody (MBL)

• Validation Controls:

  • Blocking peptide: PEP-0906 can be used with PA5-20792 for specificity validation

  • Knockdown controls: Compare antibody signal in Tmem214 knockdown versus control cells

  • Overexpression controls: Detect increased signal in cells overexpressing Tmem214

These methods provide complementary approaches for detecting Tmem214 expression, localization, and interactions, enabling comprehensive characterization in various experimental contexts.