Recombinant Human Transmembrane and coiled-coil domains protein 3 (TMCC3)

What is the structure and functional organization of TMCC3?

TMCC3 belongs to the transmembrane and coiled-coil domain family that includes TMCC1-3. The protein contains two distinctive domains: two coiled-coil domains located in the N-terminal region and two transmembrane domains in the C-terminal region . Sequence alignment analysis of TMCC3 across various vertebrate species reveals several highly conserved regions, suggesting evolutionary importance of this protein .

TMCC3 is predicted to function as an integral membrane protein localized primarily in the endoplasmic reticulum (ER) . The conservation of these domains across species indicates their functional significance, though the precise mechanism of action is still being elucidated through ongoing research.

How does TMCC3 form oligomeric structures?

TMCC3 has been demonstrated to form oligomeric structures, primarily through self-interaction. Western blotting results have revealed that full-length TMCC3 and TMCC3-Δ1 (a deletion mutant) form protein bands with molecular weights approximately three times greater than predicted (approximately 159 kDa compared to the predicted 53 kDa), strongly suggesting trimerization . In contrast, TMCC3-Δ2 (another deletion mutant) appears to form dimers, as evidenced by protein bands with approximately two-fold greater molecular weight than predicted .

Mass spectrometry analysis of proteins co-immunoprecipitated with TMCC3 has confirmed that TMCC3 primarily interacts with other TMCC3 proteins to form trimers and possibly hexamers, based on the molecular weights of identified protein bands . This oligomerization property may be critical for TMCC3's cellular functions and regulatory roles.

What protein-protein interactions are known for TMCC3?

Two key protein interactions have been characterized for TMCC3:

• Self-association: As mentioned above, TMCC3 primarily interacts with other TMCC3 proteins to form homotrimers and possibly higher-order oligomers such as hexamers .

• 14-3-3 proteins: Mass spectrometry analysis has identified that TMCC3 associates with 14-3-3 proteins, which are known regulators of intracellular signaling through their interactions with various proteins . This interaction suggests potential regulatory mechanisms where 14-3-3 may modulate TMCC3 function or vice versa.

• AKT interaction: TMCC3 has been shown to interact directly with AKT through its 1-153 amino acid domain, as demonstrated by cell-free biochemical assays in vitro and co-immunoprecipitation studies in vivo . This interaction is functionally significant as it contributes to AKT activation and subsequent effects on cell properties.

How is TMCC3 expression regulated in normal versus cancer cells?

In breast cancer models, TMCC3 shows differential expression between cancer stem cells and non-stem cancer cells. Studies using patient-derived xenograft (PDX) models of breast cancer have demonstrated significantly higher TMCC3 expression in breast cancer stem cells (BCSCs) compared to non-BCSCs . Western blotting analysis revealed that TMCC3 protein levels were higher in BCSCs than in non-BCSCs by 10.7, 9.8, and 8.9-fold for the BC0145, BC0350R1, and BC0634 tumor models, respectively .

Additionally, TMCC3 expression is elevated in mammosphere cultures (which enrich for cancer stem cells) compared to standard monolayer cultures. The protein expression was greater in mammosphere-cultured cells by 1.5, 3.8, 6.0, and 3.3-fold in MCF7, MDA-MB231, AS-B145, and AS-B634 cell lines, respectively . This consistent upregulation across multiple cell lines suggests that TMCC3 may play a fundamental role in maintaining cancer stem cell properties.

What methods are most effective for detecting TMCC3 in experimental samples?

Several complementary methods have proven effective for detecting TMCC3 in research settings:

• Western Blotting: Standard western blotting using anti-TMCC3 antibodies can effectively detect both endogenous and recombinant TMCC3. This method allows visualization of both monomeric and oligomeric forms of the protein .

• Immunohistochemistry (IHC): IHC staining has been successfully used to assess TMCC3 expression in primary tumors and metastatic lesions, allowing for spatial distribution analysis and histoscoring of expression levels .

• Flow Cytometry: For detecting TMCC3 in specific cell populations, flow cytometry using fluorescently-labeled anti-TMCC3 antibodies can be combined with other markers (such as CD44 for cancer stem cells) to identify TMCC3-expressing subpopulations .

• qRT-PCR: Quantitative reverse transcription PCR effectively measures TMCC3 mRNA expression levels and has been used in clinical studies involving 202 breast cancer specimens to correlate expression with patient outcomes .

For recombinant TMCC3, detection can be facilitated by epitope tagging (e.g., Myc-tag), which enables detection using commercially available anti-tag antibodies .

How does TMCC3 contribute to cancer stem cell maintenance?

TMCC3 plays a crucial role in maintaining cancer stem cell properties through several mechanisms:

• Self-renewal capacity: TMCC3 silencing significantly reduces mammosphere formation in breast cancer cells. In studies with AS-B145 and AS-B634 cells, TMCC3 knockdown reduced mammosphere numbers to nearly zero compared to control cells (7.4 ± 1.5 and 18.5 ± 0.8, respectively) . Conversely, TMCC3 overexpression in MCF7 cells increased mammosphere formation from 22.5 ± 2.9 to 33.5 ± 4.5 .

• ALDH activity regulation: Aldehyde dehydrogenase (ALDH) activity, a marker of cancer stem cells, is significantly reduced upon TMCC3 silencing. TMCC3 knockdown decreased the ALDH-positive population from 2.52 ± 0.96% to 0.16 ± 0.03% and 0.13 ± 0.05% in AS-B145 cells, and from 11.14 ± 3.38% to 0.94 ± 0.38% and 1.1 ± 0.38% in AS-B634 cells .

• Cancer stem cell marker expression: TMCC3 overexpression in MCF7 cells increases the CD24−CD44+ population (a marker for breast cancer stem cells) from 26.15 ± 4.38% to 48.17 ± 5.46% .

• In vivo cancer stem cell maintenance: Analysis of TMCC3-silenced tumors showed reduction of the breast cancer stem cell population (H2kd−CD24−CD44+) from 45.8% in control tumors to 15.1% in TMCC3-silenced tumors .

What is the relationship between TMCC3 and tumor metastasis?

TMCC3 demonstrates a strong association with metastatic potential in breast cancer models:

• Increased expression in metastatic lesions: TMCC3 expression is significantly higher in metastatic sites compared to primary tumors. In the BC0145 PDX model, the CD44+TMCC3+ cell subpopulation was 2.72 ± 0.7-fold higher in metastatic lymph nodes compared to primary tumors . Similarly, in the BC0634 model, histoscoring revealed highest TMCC3 expression in lung metastases, followed by lymph node metastases, and then primary tumors .

• Migration promotion: TMCC3 silencing significantly reduces cancer cell migration in vitro. Knockdown of TMCC3 in AS-B145 and AS-B634 cells reduced migrated cell numbers to 1.19 ± 0.03 and 1.02 ± 0.03 (×1000) cells/well, respectively, compared to 2.72 ± 0.26 and 3.03 ± 0.35 (×1000) cells/well in control cells .

• Metastasis inhibition in vivo: TMCC3 silencing completely abolished metastasis in animal models. In control AS-B634 tumors, lymph node and lung metastasis frequencies were 75% (3/4) and 50% (2/4), respectively, while no metastasis was observed in TMCC3-knockdown tumors .

These findings strongly suggest that TMCC3 is a critical factor in promoting cancer metastasis, making it a potential therapeutic target for preventing metastatic spread.

How does TMCC3 interact with the AKT signaling pathway?

TMCC3 plays a significant role in AKT activation through direct interaction rather than through upstream pathway components:

• Direct binding to AKT: TMCC3 has been demonstrated to interact directly with AKT through its 1-153 amino acid domain, as confirmed by cell-free biochemical assays, co-immunoprecipitation, and interaction domain mapping studies .

• AKT phosphorylation promotion: TMCC3 knockdown in AS-B634 cells decreases the relative levels of pAKT S473/AKT to 0.4 and 0.2, and pAKT T308/AKT to 0.6 and 0.3 with two different shRNA constructs . Conversely, TMCC3 overexpression in MCF7 cells increases pAKT S473 and pAKT T308 levels to 1.3 and 1.5-fold, respectively, upon insulin stimulation .

• Mechanism independent of PI3K/PDK1: Importantly, TMCC3-induced AKT activation appears to be independent of upstream PI3K or PDK1 phosphorylation. Neither TMCC3 silencing nor overexpression affects the phosphorylation of the regulatory subunit p85 of PI3K or PDK1 . Additionally, TMCC3 overexpression does not enhance membrane translocation of the catalytic subunit p110-α of PI3K .

This direct interaction with AKT represents a novel mechanism by which TMCC3 can influence cellular signaling and phenotypes, particularly in cancer stem cells.

What functional domains of TMCC3 are critical for its biological activity?

Domain truncation studies have identified specific regions of TMCC3 that are essential for its function:

• AKT-interacting domain (amino acids 1-153): This domain is critical for TMCC3's ability to interact with and activate AKT. Domain mapping studies have shown that this region is essential for TMCC3-induced AKT activation, self-renewal, and metastasis .

• Coiled-coil domains: Located in the N-terminal region, these domains likely play a role in protein-protein interactions, particularly in the formation of TMCC3 trimers. Deletion mutants lacking these domains show altered oligomerization patterns .

• Transmembrane domains: Located in the C-terminal region, these domains are responsible for anchoring TMCC3 to cellular membranes, particularly the endoplasmic reticulum. They may also contribute to protein stability and localization .

Understanding these domain-specific functions provides opportunities for targeted interventions that could disrupt specific TMCC3 activities while preserving others.

What are effective strategies for silencing or overexpressing TMCC3 in experimental models?

Several approaches have been successfully employed to manipulate TMCC3 expression:

• Lentivirus-mediated shRNA silencing: This approach has proven highly effective for TMCC3 knockdown. Using shRNA clones #A, #B, and #C, TMCC3 mRNA levels were reduced to 0.24 ± 0.02, 0.09 ± 0.02, and 0.3 ± 0.01 folds, respectively, of control in AS-B145 cells; and to 0.42 ± 0.02, 0.23 ± 0.06, and 0.82 ± 0.12 folds, respectively, in AS-B634 cells . Western blotting confirmed the reduction of TMCC3 protein to negligible levels .

• Lentivirus-mediated overexpression: Stable overexpression of TMCC3 has been achieved in cell lines with low endogenous TMCC3 expression, such as MCF7 . This approach is useful for gain-of-function studies.

• Expression of deletion mutants: Creation of domain-specific deletion mutants (TMCC3-Δ1, TMCC3-Δ2) has been effective for studying the role of specific TMCC3 domains in oligomerization and function .

• Recombinant expression in HEK293 cells: For producing recombinant TMCC3 protein, expression in HEK293 cells with epitope tagging (e.g., Myc-tag) has been successful .

These approaches provide a toolkit for investigating TMCC3 function through both loss-of-function and gain-of-function experimental designs.

What assays are most informative for studying TMCC3's role in cancer stem cell properties?

Several functional assays have proven valuable for assessing TMCC3's impact on cancer stem cell properties:

• Mammosphere formation assay: This in vitro assay measures self-renewal capacity, a key property of cancer stem cells. Changes in mammosphere number and size upon TMCC3 manipulation provide quantitative measures of stemness .

• ALDH activity assay: Measurement of aldehyde dehydrogenase activity using the ALDEFLUOR assay is an established method for identifying and quantifying cancer stem cells. Flow cytometry analysis of ALDH-positive populations can assess the impact of TMCC3 on this stem cell marker .

• Flow cytometry for stem cell markers: Analysis of established stem cell markers (e.g., CD24−CD44+ for breast cancer) by flow cytometry allows quantification of cancer stem cell populations upon TMCC3 manipulation .

• In vivo limiting dilution assay: This gold-standard assay determines tumor-initiating capacity by injecting serial dilutions of cells into immunodeficient mice and calculating the frequency of tumor-initiating cells. This approach has revealed that TMCC3 silencing significantly reduces tumor-initiating capacity (from 1:19,707 to 1:70,682 in AS-B145 and from 1:59 to 1:987 in AS-B634) .

• Migration assay: As metastasis is a key property of aggressive cancer stem cells, in vitro migration assays provide insights into TMCC3's role in this process .

These complementary assays together provide a comprehensive assessment of TMCC3's impact on cancer stem cell properties in both in vitro and in vivo settings.

How does TMCC3 expression correlate with clinical outcomes in cancer patients?

Clinical studies have established strong correlations between TMCC3 expression and patient outcomes:

• Prognostic significance: In a study of 202 breast cancer specimens, qRT-PCR analysis demonstrated that higher TMCC3 mRNA expression correlated with poorer clinical outcomes, including in early-stage breast cancer .

• Independent prognostic factor: Multivariable analysis identified TMCC3 expression as an independent risk factor for survival in breast cancer patients, highlighting its potential value as a prognostic biomarker .

These clinical correlations, combined with the functional evidence for TMCC3's role in cancer stem cell maintenance and metastasis, suggest that TMCC3 may serve as both a biomarker for aggressive disease and a potential therapeutic target in cancer treatment.

What are the methodological considerations for producing recombinant TMCC3 for research applications?

For researchers aiming to produce recombinant TMCC3 for in vitro studies, several methodological considerations are important:

• Expression system selection: HEK293 cells have been successfully used for recombinant TMCC3 expression . This mammalian expression system likely provides appropriate post-translational modifications and protein folding for TMCC3.

• Epitope tagging: Addition of epitope tags (such as Myc-tag) facilitates detection and purification of recombinant TMCC3 . The tag positioning should be carefully considered to avoid interference with functional domains.

• Domain-specific constructs: For studying specific functions, expression of domain-deletion mutants can be informative. Previous studies have successfully expressed TMCC3-Δ1 and TMCC3-Δ2 deletion mutants .

• Oligomerization consideration: Given that TMCC3 naturally forms trimers and possibly higher-order oligomers, purification and analysis methods should account for these oligomeric states . Size exclusion chromatography may be useful for separating different oligomeric forms.

• Interaction partner co-expression: For studying TMCC3 interactions with partners like AKT or 14-3-3 proteins, co-expression or in vitro reconstitution approaches may be necessary .

These methodological considerations can help researchers design appropriate strategies for producing functional recombinant TMCC3 for various research applications.