Recombinant Human Transmembrane and coiled-coil domains protein 1 (TMCC1)

What is the basic structure and localization of TMCC1?

TMCC1 is a member of the transmembrane and coiled-coil domain family that includes at least three proteins (TMCC1-3) conserved from nematode to human. Structurally, TMCC1 contains two adjacent transmembrane domains near the C-terminus, along with coiled-coil domains . The protein localizes specifically to the rough endoplasmic reticulum through its C-terminal transmembrane domains (aa 571-653), while its N-terminal region and C-terminal tail reside in the cytoplasm . This unique topology allows TMCC1 to interact with both cytosolic proteins and participate in membrane organization functions.

Experimental research has confirmed that each transmembrane domain (TMCC1(571-615) and TMCC1(615-653)) possesses ER-targeting properties independently, suggesting functional redundancy in the localization mechanism . When studying TMCC1 localization, immunofluorescence with established ER markers such as calnexin shows clear colocalization patterns, providing a reliable method for confirming proper expression of recombinant TMCC1 constructs .

What physiological functions has TMCC1 been associated with?

TMCC1 plays a significant role in endosome fission, a process where endosomes split into smaller vesicles. Specifically, TMCC1 promotes this fission by mediating contact between the ER and endosomes . This process is crucial for sorting cellular material into either degradation pathways (via lysosomes) or recycling pathways (back to the cell surface) . Additionally, TMCC1 has been implicated in ER organization, as overexpression studies demonstrate that altered TMCC1 levels can lead to structural deformations in the ER network .

Recent research has also identified a circular RNA form of TMCC1 (circTmcc1) that contributes to glutamate metabolism in astrocytes and regulates spatial memory formation by mediating neuronal synaptic plasticity in hepatic encephalopathy models . This indicates that TMCC1, across its various forms, may have broader neurophysiological functions than initially appreciated.

What diseases have been associated with TMCC1 dysfunction?

TMCC1 has been linked to several pathological conditions. These include laryngotracheoesophageal cleft, type 2 diabetes mellitus, nonpapillary renal cell carcinoma, general cancer pathways, and glioblastoma . The involvement in these diverse conditions suggests TMCC1 may influence multiple cellular processes beyond its known ER and endosomal functions.

In neurological contexts, studies indicate that the circular form of TMCC1 (circTmcc1) has regulatory effects on astrocyte function and neuronal synaptic plasticity, with implications for cognitive function in hepatic encephalopathy . Specifically, circTmcc1 contributes to the regulation of glutamate metabolism and can modulate spatial memory through mechanisms involving NF-κB and CREB signaling pathways .

What are the optimal expression systems for producing recombinant human TMCC1?

When expressing recombinant human TMCC1, researchers should carefully consider the expression system based on downstream applications. For structural studies requiring properly folded and post-translationally modified protein, mammalian expression systems such as HEK293 or CHO cells are recommended, as they have the appropriate ER machinery to handle membrane proteins .

Recommended transfection protocol parameters:

• For HeLa cells: 2 × 10⁴ cells per cm² cultured in DMEM with 5-10% FBS

• For optimal detection: GFP or FLAG-tagged constructs can be used for visualization

• Expression time: 24-48 hours post-transfection for optimal balance between expression and minimal ER deformation

How can researchers effectively study TMCC1 protein interactions?

TMCC1 forms homo- or hetero-dimers/oligomers with other TMCC proteins through its coiled-coil domains and interacts with ribosomal proteins via its cytosolic region . To study these interactions, researchers should consider:

• Co-immunoprecipitation assays: Using anti-TMCC1 antibodies to pull down protein complexes from cell lysates. Specificity can be verified using TMCC1 siRNA knockdown controls, which should show >80% reduction in the detected protein .

• Sucrose gradient fractionation: This technique can separate rough ER components containing TMCC1 from other cellular compartments. In these experiments, TMCC1 co-fractionates with rough ER protein CLIMP-63 and ribosomal protein RPL4 in the bottom layers of sucrose gradients .

• Prediction tools for RNA-protein interactions: For studying potential interactions between circTmcc1 and transcriptional regulators, computational tools like RPISeq can be valuable. This approach combines random forest (RF) classifiers and support vector machine (SVM) classifiers to predict interaction probabilities .

• Protein interaction network analysis: Tools such as STRING can be used to establish and visualize potential interaction networks between TMCC1 and other proteins, which can be further classified using kmeans clustering into functional groups .

What methodologies are most effective for studying TMCC1's role in ER organization?

To investigate TMCC1's functions in ER organization, researchers can employ several complementary approaches:

• Overexpression studies: Transfecting cells with GFP-TMCC1 or specific domains (particularly the transmembrane domains) at varying expression levels. At high expression levels, both full-length TMCC1 and its transmembrane domains cause ER deformation, visible by co-staining with ER markers like calnexin .

• Domain mapping: Expressing truncated constructs to identify functional domains. Research has shown that TMCC1(571-653) is sufficient for ER targeting, while TMCC1(1-575) localizes to the cytosol . This approach helps delineate which protein regions are responsible for specific functions.

• Knockout/knockdown experiments: Using siRNA targeting TMCC1 (achieving >80% knockdown efficiency) allows researchers to study loss-of-function effects on ER morphology and associated processes .

• Live cell imaging: For studying dynamic ER remodeling processes, time-lapse microscopy of fluorescently tagged TMCC1 constructs can reveal real-time effects on ER structure and membrane contacts with other organelles.

How is TMCC1 expression regulated at the transcriptional level?

While the search results don't provide comprehensive information on TMCC1 transcriptional regulation, studies on circTmcc1 offer insights into regulatory mechanisms that may be relevant. Research shows that circTmcc1 interacts with the NF-κB p65-CREB transcriptional complex, regulating expression of the astrocyte transporter EAAT2 . This suggests that similar transcriptional machinery may influence TMCC1 expression.

For researchers investigating transcriptional regulation of TMCC1, computational tools can identify potential transcription factors. Tools like BART and ChEA3 have been used to predict transcriptional factors involved in circTmcc1-mediated gene regulation, ranking candidates based on Irwin-Hall p-value and Integrated Scale Rank metrics . These approaches could be applied to study TMCC1 regulation directly.

When designing experiments to study TMCC1 transcriptional regulation, researchers should consider:

• Promoter analysis using luciferase reporter assays

• Chromatin immunoprecipitation (ChIP) to identify transcription factor binding sites

• RNA sequencing after knockdown/overexpression of candidate transcription factors

What is known about circular RNA forms of TMCC1 and their functional significance?

A circular RNA form of TMCC1 (circTmcc1) has been identified with significant regulatory functions distinct from the protein-coding counterpart. Research demonstrates that circTmcc1 improves astrocytic glutamate metabolism and spatial memory via NF-κB and CREB signaling pathways in models of hepatic encephalopathy .

In bile duct ligation (BDL) mouse models, circTmcc1 has been shown to:

• Bind with the NF-κB p65-CREB transcriptional complex

• Regulate expression of the astrocyte transporter EAAT2

• Contribute to secretion of proinflammatory mediators

• Modulate glutamate metabolism in astrocytes

• Improve spatial memory by mediating neuronal synaptic plasticity

For researchers studying circTmcc1, RNA sequencing approaches are essential. Methods involve filtering reads using FastQc, trimming low-quality reads with Trimmomatic, and aligning with STAR aligner to the mouse genome (mm10) . Expression changes can be quantified using FPKM calculations with Cuffnorm or through alternative approaches using Salmon quantifier and edgeR .

How does TMCC1 contribute to disease pathology in cancer and neurological conditions?

TMCC1 has been associated with several cancer types, including nonpapillary renal cell carcinoma and glioblastoma . Although the precise mechanisms remain to be fully elucidated, TMCC1's role in ER organization and membrane dynamics may influence cancer cell survival, proliferation, or metastasis pathways.

In neurological contexts, research on circTmcc1 provides insights into potential neuropathophysiological mechanisms. In hepatic encephalopathy models, circTmcc1 modulates astrocyte function and glutamate metabolism, with downstream effects on neural synaptic plasticity and spatial memory . These findings suggest that TMCC1 and its circular RNA form may influence neurological function through regulation of astrocyte-neuron interactions and glutamatergic signaling pathways.

For researchers investigating disease contributions, comparing TMCC1 expression and function in normal versus diseased tissues using techniques such as immunohistochemistry, Western blotting, and functional assays would be valuable approaches.

What experimental models are available for studying TMCC1 in disease contexts?

Several experimental models have been developed and validated for studying TMCC1 in disease contexts:

• Cell culture models: Multiple human cell lines express TMCC1, making them suitable for in vitro studies . For modeling hyperammonemic conditions relevant to hepatic encephalopathy, treatment protocols using 20 mM NH₄Cl for 24 hours have been established .

• Mouse models: The bile duct ligation (BDL) mouse model has been specifically validated for studying circTmcc1 in hepatic encephalopathy . This model demonstrates specific alterations in circTmcc1 expression in the brain cortex.

• Astrocyte culture systems: C8-D1a cells (2 × 10⁴ cells per cm²) cultured in DMEM with 10% FBS provide a reliable in vitro system for studying TMCC1/circTmcc1 functions in astrocytes .

When designing experiments using these models, researchers should include appropriate controls and validate their findings using multiple complementary approaches, as single methodologies may not capture the full complexity of TMCC1's functions in disease states.

What bioinformatic approaches are recommended for analyzing TMCC1 expression data?

For comprehensive analysis of TMCC1 expression data, researchers should implement a multi-tool bioinformatic pipeline:

• Quality control and preprocessing: Use FastQc for checking RNA sequencing read quality, and Trimmomatic for trimming low-quality reads .

• Alignment and quantification: Employ either STAR aligner for genome alignment followed by Cuffnorm for FPKM calculations, or use Salmon quantifier for transcript level quantification .

• Differential expression analysis: Apply statistical methods such as Student's t-test or edgeR for identifying significantly altered expression. Filtering criteria should exclude genes with an average FPKM value less than 1 and select genes with log2 value differences of more than 0.5 or less than -0.5 between control and experimental groups .

• Functional prediction: For protein interaction studies, tools like STRING can classify interaction networks using kmeans clustering into functional clusters . For RNA-protein interactions, RPISeq provides interaction probability scores using RF and SVM classifiers .

These bioinformatic approaches provide a robust framework for analyzing TMCC1 expression and interactions in various experimental settings.

What are the optimal methods for detecting endogenous versus recombinant TMCC1 in experimental systems?

For reliable detection of TMCC1 in experimental systems, researchers have several validated options:

• Western blotting: Custom antibodies raised against the N-terminal fragment TMCC1(1-200) have successfully detected endogenous TMCC1 in whole cell extracts . This region was chosen specifically because it is unique among TMCC family members in humans. Western blotting can detect protein bands with molecular weights corresponding to the theoretical molecular weight of TMCC1, with specificity confirmed using siRNA knockdown controls .

• Immunofluorescence: For localization studies, antibodies against TMCC1 combined with ER markers like calnexin provide clear visualization of protein distribution . This approach is particularly useful for studying colocalization with other cellular components.

• Fluorescent tagging: For recombinant protein studies, GFP-TMCC1 constructs expressed at low levels show reliable localization throughout the ER, with distribution patterns similar to endogenous protein . FLAG-tagged constructs provide an alternative when GFP fusion might interfere with function .

• Subcellular fractionation: Sucrose gradient fractionation techniques can separate rough ER components containing TMCC1 from other cellular compartments, providing an additional method for confirming localization and studying protein complexes .

When comparing endogenous versus recombinant protein, researchers should be mindful that high expression levels of recombinant TMCC1 can cause ER deformation, potentially complicating interpretation of results .