Recombinant Mouse Transmembrane and coiled-coil domains protein 3 (Tmcc3)

What is the basic structure of TMCC3 protein?

TMCC3 (Transmembrane and coiled-coil domain family 3) contains two coiled-coil domains and two transmembrane domains. The coiled-coil regions mediate protein-protein interactions and oligomerization, while the transmembrane domains anchor the protein to cellular membranes, particularly the endoplasmic reticulum (ER). Recombinant TMCC3 has a predicted molecular weight of approximately 53 kDa, though oligomeric forms with much higher molecular weights have been observed in experimental systems . These structural features are critical for TMCC3's cellular functions and interactions with other proteins.

Where is TMCC3 localized within cells?

Immunostaining and confocal microscopy studies have demonstrated that TMCC3 primarily localizes to the endoplasmic reticulum through its transmembrane domains . Cell fractionation experiments have shown that TMCC3 proteins are present in the nuclear membrane fraction, with full-length TMCC3 also detected in the cytosolic fraction . This localization pattern is similar to that observed for TMCC1, suggesting conserved subcellular distribution among TMCC family members. The ER localization is particularly important for understanding TMCC3's potential roles in cellular processes like protein synthesis, folding, and quality control.

What is the tissue distribution of TMCC3 in normal mammalian tissues?

TMCC3 shows tissue-specific expression patterns in mammals. In humans, highest expression levels are found in whole brain, testis, and spinal cord compared to other tissues . This suggests specialized functions in neural and reproductive systems. In developing mouse embryos, TMCC3 shows dynamic expression patterns with strong signals in the mesenchyme of developing tongue at embryonic day 12 (E12), and in trigeminal ganglion, oral epithelium, hindbrain, lung, kidney, and somites at E14 . These tissue-specific expression patterns provide important clues about potential physiological functions of TMCC3 in different organ systems.

How is TMCC3 expression altered in cancer?

TMCC3 expression is significantly altered in cancer contexts. In breast cancer, several key changes have been documented:

• Higher expression in breast cancer stem cells (BCSCs) compared to non-BCSCs .

• Increased expression in mammosphere-cultured cells compared to monolayer-cultured cells, with 1.5-fold increase in MCF7, 3.8-fold in MDA-MB231, 6.0-fold in AS-B145, and 3.3-fold in AS-B634 cell lines .

• Elevated expression in metastatic lesions (lymph nodes and lungs) compared to primary tumors .

• Clinical analysis of 202 breast cancer specimens showed that higher TMCC3 expression correlates with poorer clinical outcomes, with multivariable analysis identifying TMCC3 expression as an independent risk factor for survival .

These expression changes suggest TMCC3 plays important roles in cancer progression, particularly in maintaining cancer stem cell properties and promoting metastasis.

What techniques are most reliable for detecting TMCC3 expression in tissue samples?

Several complementary techniques have proven effective for analyzing TMCC3 expression:

• In situ hybridization: Effective for examining spatial expression patterns in developing tissues during embryogenesis. This method has successfully revealed TMCC3 expression in specific structures like developing hindbrain, tongue mesenchyme, and trigeminal ganglion .

• Immunohistochemistry (IHC): Valuable for analyzing TMCC3 protein expression in primary tumors and metastatic lesions, with quantification using histoscores (h-score) to compare expression levels between different tissue samples .

• Western blotting: Reliable for detecting TMCC3 protein expression levels and oligomerization states in cell and tissue lysates, particularly when combined with appropriate controls .

• Quantitative RT-PCR (qRT-PCR): Effective for measuring TMCC3 mRNA expression in clinical specimens and correlating expression levels with clinical outcomes .

• Flow cytometry: Useful for analyzing TMCC3 expression in specific cell populations, particularly when combined with other markers like CD44 or ALDH to identify cancer stem cells .

How can TMCC3 expression be effectively manipulated in experimental models?

Based on published methodologies, several approaches have proven successful for manipulating TMCC3 expression:

• RNA interference (RNAi): Short hairpin RNAs (shRNAs) targeting TMCC3 have been used to silence expression in cancer cell lines. For example, shTM #A and shTM #B constructs effectively reduced TMCC3 expression in AS-B145 and AS-B634 cells .

• Overexpression systems: Stable transfection of TMCC3 expression constructs (with appropriate tags like Myc) has been effective for studying gain-of-function effects, as demonstrated in MCF7 cells and HEK293 cells .

• Domain deletion mutants: Constructs lacking specific domains (e.g., TMCC3-Δ1 lacking the first coiled-coil domain, TMCC3-Δ2 lacking both coiled-coil domains) have been valuable for dissecting domain-specific functions .

• Patient-derived xenograft (PDX) models: These provide clinically relevant systems for studying TMCC3 expression and function in vivo, particularly for cancer-related studies .

What evidence supports TMCC3 as a marker for cancer stem cells?

Multiple lines of experimental evidence support TMCC3 as a marker for cancer stem cells (CSCs):

• Functional assays: TMCC3 silencing significantly reduced mammosphere formation capacity in breast cancer cell lines (AS-B145 and AS-B634), while TMCC3 overexpression in MCF7 cells enhanced mammosphere formation. Specifically, mammosphere numbers were reduced from 7.4±1.5 to 1.0±0.4 (shTM #A) and 0.2±0.2 (shTM #B) in AS-B145 cells .

• Stem cell marker expression: TMCC3 knockdown reduced the ALDH+ subpopulation (a known CSC marker) in AS-B145 cells from 33.7% to 22.9% (shTM #A) and 16.8% (shTM #B). Conversely, TMCC3 overexpression in MCF7 cells increased the CD24-CD44+ subset from 26.15±4.38% to 48.17±5.46% .

• Tumor-initiating capacity: In vivo studies demonstrated that TMCC3 knockdown reduced tumor-initiating capacity (TIC) of breast cancer cells. TIC of AS-B145 decreased from 1:19,707 to 1:70,682 (p=0.014) after TMCC3 knockdown .

• Expression in metastatic lesions: Higher proportions of CD44+TMCC3+ cells were found in metastatic lymph nodes (15.3%) compared to primary tumors (4.1%), suggesting enrichment of TMCC3-expressing cells during metastasis .

These findings collectively indicate that TMCC3 plays crucial roles in maintaining cancer stem cell properties and could serve as a valuable marker for identifying these therapeutically important cell populations.

How does TMCC3 interact with the AKT signaling pathway in cancer?

TMCC3 directly interacts with and regulates the AKT signaling pathway, which is central to cancer progression:

• Direct interaction: TMCC3 interacts directly with AKT through its 1-153 amino acid domain, as demonstrated by cell-free biochemical assays in vitro and co-immunoprecipitation and interaction domain mapping assays in vivo .

• Effect on AKT phosphorylation: TMCC3 knockdown in AS-B634 cells decreased phosphorylation of AKT at both S473 and T308 residues (to 0.4 and 0.6 of control levels for shTM #A, and 0.2 and 0.3 for shTM #B, respectively). Conversely, TMCC3 overexpression in MCF7 cells increased AKT phosphorylation at these sites upon insulin stimulation (to 1.3 and 1.5-fold of control levels) .

• Specificity of effect: Importantly, TMCC3 does not appear to enhance AKT phosphorylation via activation of upstream regulators like PI3K/PDK1, as phosphorylation of the regulatory subunit p85 of PI3K and PDK1 did not change upon TMCC3 manipulation .

• Functional significance: The AKT-interacting domain of TMCC3 is essential for TMCC3-induced AKT activation, self-renewal, and metastasis, highlighting the importance of this interaction in cancer progression .

This TMCC3-AKT interaction represents a potential therapeutic target for disrupting cancer stem cell maintenance and tumor progression.

What is the relationship between TMCC3 expression and cancer metastasis?

TMCC3 appears to play a significant role in cancer metastasis, as evidenced by several experimental observations:

• Increased expression in metastatic lesions: Analysis of breast cancer PDX models showed that TMCC3 expression is higher in metastatic sites compared to primary tumors. Specifically, 15.3% of cells in metastatic lymph nodes were CD44+TMCC3+ compared to only 4.1% in primary tumors of BC0145 PDX .

• Expression gradient in metastatic progression: Immunohistochemical analysis of BC0634 PDX showed a progressive increase in TMCC3 expression from primary tumors to lymph node metastases to lung metastases, as quantified by histoscores .

• Enrichment during metastasis: The CD44+TMCC3+ subsets in metastatic lymph nodes showed a 2.72±0.7-fold increase compared to primary tumor cells, suggesting selective advantage of TMCC3-expressing cells during metastatic spread .

These findings suggest that TMCC3 may contribute to metastatic processes through its role in maintaining cancer stem cell properties, potentially enhancing cellular capabilities for invasion, migration, and colonization of distant sites.

How does TMCC3

oligomerization occur and what domains are essential? TMCC3 oligomerization is a complex process involving multiple protein domains with distinct contributions:

• Trimer formation: Full-length TMCC3 and TMCC3-Δ1 (lacking the first coiled-coil domain) form trimers, as evidenced by protein bands with molecular weights approximately three-fold greater than the predicted monomeric weight on Western blots .

• Role of coiled-coil domains: TMCC3-Δ2 (lacking both coiled-coil domains) forms dimers rather than trimers, indicating that the second coiled-coil domain is critical for proper trimer formation .

• Contribution of transmembrane domains: The transmembrane domains (present in all constructs including TMCC3-Δ2) can mediate protein-protein interactions between TMCC3 proteins, but these interactions differ from those facilitated by full-length protein .

• Higher-order complexes: Mass spectrometry analysis of immunoprecipitated TMCC3 revealed protein bands with molecular weights much greater than 250 kDa, suggesting formation of higher-order oligomers beyond trimers, possibly hexamers .

These findings establish a model where the second coiled-coil domain directs proper trimerization, while the transmembrane domains provide additional interaction surfaces that contribute to oligomer formation and stability.

What proteins interact with TMCC3 and what are the functional implications?

Based on experimental evidence, TMCC3 interacts with several proteins that suggest important functional roles:

• Self-association: TMCC3 proteins interact with each other to form oligomers, primarily trimers and possibly larger complexes .

• 14-3-3 proteins: Mass spectrometry analysis of co-immunoprecipitated proteins revealed that TMCC3 associates with 14-3-3 proteins, which are known to regulate intracellular signaling by interacting with phosphorylated motifs on target proteins . This suggests potential regulation of TMCC3 function through phosphorylation-dependent mechanisms.

• AKT: TMCC3 interacts directly with AKT through its 1-153 amino acid domain . This interaction is functionally significant, as it promotes AKT phosphorylation and activation, which in turn supports cancer stem cell properties and metastasis.

Unlike TMCC1, which has been reported to interact with ribosomal proteins (L4, L6, and P0), current evidence does not indicate that TMCC3 interacts with ribosomal proteins . This suggests potentially divergent functions between TMCC family members despite structural similarities.

What is the expression pattern of TMCC3 during mouse embryonic development?

TMCC3 exhibits a dynamic and tissue-specific expression pattern during mouse embryonic development:

• At embryonic day 12 (E12):

  • Strongest expression in the mesenchyme of developing tongue

  • Broad but weak expression in developing hindbrain and lung-forming tissues

• At embryonic day 14 (E14):

  • Strong expression in the trigeminal ganglion, oral epithelium, and hindbrain-forming tissues

  • Clear positive expression in developing lung, kidney, and somites

This spatiotemporal expression pattern suggests potential roles for TMCC3 in the development of neural tissues, orofacial structures, respiratory and urinary systems, and musculoskeletal structures. The shift in expression patterns between E12 and E14 indicates developmental stage-specific functions that may relate to tissue differentiation and organogenesis.

What are the implications of TMCC3's developmental expression for understanding its function?

The developmental expression pattern of TMCC3 provides several insights into its potential physiological functions:

• Neural development: Strong expression in hindbrain and trigeminal ganglion correlates with high expression in adult human brain and spinal cord, suggesting conserved functions in neural tissues throughout life .

• Epithelial-mesenchymal interactions: Expression in both epithelial (oral epithelium) and mesenchymal tissues (tongue mesenchyme) suggests potential involvement in epithelial-mesenchymal interactions critical for proper organogenesis .

• Tissue differentiation: Expression in developing somites may indicate roles in musculoskeletal differentiation, while expression in developing lung and kidney suggests functions in branching morphogenesis common to these organs .

• Regulatory functions: The association of TMCC3 with 14-3-3 proteins and its role in AKT signaling suggest that TMCC3 may participate in signaling pathways important for developmental processes, potentially serving as a scaffold or regulatory protein.

Understanding these developmental functions may provide insights into how dysregulation of TMCC3 contributes to disease states such as cancer, where developmental programs are often aberrantly reactivated.

How can deletion mutants be designed to study domain-specific functions of TMCC3?

Based on successful approaches in the literature, an effective strategy for creating TMCC3 deletion mutants includes:

• Domain-specific deletions: Design constructs that systematically remove functional domains:

  • TMCC3-Δ1: Deletion of the first coiled-coil region

  • TMCC3-Δ2: Deletion of both coiled-coil regions, retaining only the transmembrane domains

• Expression vector selection: Clone constructs into mammalian expression vectors with appropriate tags (e.g., Myc-tag) for detection and purification .

• Verification of expression: Confirm proper expression and expected molecular weight of mutant proteins through Western blotting .

• Oligomerization analysis: Examine how domain deletions affect protein-protein interactions and oligomerization by analyzing apparent molecular weights under non-reducing conditions .

• Localization studies: Determine whether deletions affect subcellular localization using immunofluorescence and confocal microscopy .

• Functional assessments: Evaluate how domain deletions impact protein function through appropriate assays, such as effects on AKT activation or cancer stem cell properties .

This systematic approach has revealed critical insights, such as the importance of the second coiled-coil domain for trimer formation and the role of transmembrane domains in protein localization .

What experimental approaches are effective for studying TMCC3-protein interactions?

Several complementary techniques have proven effective for studying TMCC3 interactions with other proteins:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged TMCC3 (e.g., Myc-tagged) to pull down TMCC3 and its interacting partners, followed by Western blotting or mass spectrometry analysis .

• Mass spectrometry: Analysis of co-immunoprecipitated proteins to identify novel interaction partners, as demonstrated by the discovery of 14-3-3 proteins as TMCC3 interactors .

• Domain mapping: Using deletion mutants to identify specific regions required for protein interactions, as shown for the AKT-interacting domain (amino acids 1-153) of TMCC3 .

• Cell-free biochemical assays: In vitro binding assays using purified proteins to confirm direct interactions, as used to demonstrate TMCC3-AKT binding .

• Cellular validation: Confirming interactions identified through biochemical methods in cellular contexts, using techniques like fluorescence co-localization or proximity ligation assays.

These approaches have successfully identified important TMCC3 interactions, including self-association, binding to 14-3-3 proteins, and direct interaction with AKT .

How does TMCC3 expression correlate with clinical outcomes in cancer?

TMCC3 expression has significant clinical correlations in breast cancer:

• Prognostic value: Higher TMCC3 mRNA expression correlates with poorer clinical outcomes in breast cancer patients, as demonstrated by analysis of 202 breast cancer specimens using qRT-PCR .

• Early-stage indicator: This correlation is observed even in early-stage breast cancer, suggesting TMCC3 expression may be an early indicator of aggressive disease .

• Independent prognostic factor: Multivariable analysis identified TMCC3 expression as an independent risk factor for survival, indicating its prognostic value extends beyond established clinical parameters .

• Metastatic potential: Higher TMCC3 expression in metastatic lesions compared to primary tumors suggests its potential utility as a marker for metastatic risk assessment .

These findings establish TMCC3 as a promising prognostic biomarker in breast cancer that could help identify patients who might benefit from more aggressive therapeutic approaches or targeted therapies.

What are potential therapeutic strategies targeting TMCC3?

Based on current understanding of TMCC3 biology, several therapeutic approaches could be considered:

• Direct TMCC3 inhibition: Developing small molecules or peptides that bind to TMCC3 and disrupt its function, particularly focusing on domains critical for oligomerization or protein interactions .

• Targeting TMCC3-AKT interaction: Since the AKT-interacting domain of TMCC3 (amino acids 1-153) is essential for TMCC3-induced AKT activation and cancer stem cell maintenance, this interface represents a promising therapeutic target .

• Gene silencing approaches: Given the effectiveness of shRNA-mediated TMCC3 knockdown in reducing cancer stem cell properties and tumor growth, RNA interference-based therapeutics could be developed .

• Combination with conventional therapies: TMCC3-targeted therapies might be particularly effective in combination with conventional treatments to address therapy-resistant cancer stem cells .

• Antibody-based therapies: Developing antibodies against TMCC3 could potentially target TMCC3-expressing cancer cells, particularly cancer stem cells with high TMCC3 expression.

The finding that TMCC3 is crucial for maintenance of cancer stem cell features through AKT regulation suggests that targeting this pathway could address key challenges in cancer treatment, including metastasis and therapy resistance .