Recombinant Mouse Transmembrane and coiled-coil domain-containing protein 2 (Tmco2)

What is Mouse Transmembrane and Coiled-Coil Domain-Containing Protein 2 (Tmco2)?

Mouse Tmco2 is a protein-coding gene that belongs to the transmembrane and coiled-coil domain-containing protein family. Similar to its orthologs in other species, mouse Tmco2 likely contains both transmembrane regions that anchor it within cellular membranes and coiled-coil domains that facilitate protein-protein interactions. These structural features suggest its involvement in cellular scaffolding, signaling, or transport mechanisms. The gene encodes a protein that is expressed in multiple tissues, including neuronal cells, where it may play important roles in cellular architecture and function .

How does Mouse Tmco2 compare structurally to its human ortholog?

Mouse Tmco2 shares significant structural homology with its human ortholog. Both proteins contain the characteristic transmembrane domains and coiled-coil regions that define this protein family. The human ortholog of the related TMCC2 protein has been identified as having an evolutionarily conserved interaction with the amyloid protein precursor (APP), a central protein in Alzheimer's disease pathogenesis . Given evolutionary conservation patterns in this protein family, mouse Tmco2 likely exhibits similar domain organization and potentially analogous binding partners, making it a valuable model for studying the fundamental biology of this protein class.

What is the typical expression pattern of Tmco2 in mouse tissues?

Tmco2 expression varies across mouse tissues, with notable presence in neural tissues and reproductive organs. While comprehensive expression data specifically for mouse Tmco2 is still emerging, studies of related proteins in this family suggest tissue-specific expression patterns. Based on conservation of function between species, Tmco2 likely exhibits expression patterns that correlate with its biological roles. The protein may show differential expression during development and in response to various physiological stimuli, suggesting regulatory functions that are context-dependent and tissue-specific.

What are the key differences between Tmco2 and other transmembrane and coiled-coil domain proteins?

Tmco2 shares structural features with other transmembrane and coiled-coil domain proteins but has distinct sequence characteristics and likely specialized functions. The table below compares key features of mouse Tmco2 with related proteins:

How does post-translational modification affect Mouse Tmco2 function in neural tissues?

Post-translational modifications (PTMs) of mouse Tmco2 likely play crucial roles in regulating its function, localization, and protein-protein interactions in neural tissues. Research into related transmembrane proteins suggests that phosphorylation, glycosylation, and ubiquitination may modulate Tmco2's activity. When investigating Tmco2 PTMs, researchers should employ phospho-specific antibodies, mass spectrometry, and site-directed mutagenesis to identify modification sites and their functional consequences. Comparative studies between normal and pathological tissue samples can reveal alterations in Tmco2 modification patterns associated with neurological conditions, particularly given the established link between human TMCC2 and Alzheimer's disease pathology .

What are the methodological challenges in producing pure, active recombinant Mouse Tmco2?

Producing pure, active recombinant mouse Tmco2 presents several technical challenges due to its transmembrane domains, which often lead to protein aggregation and reduced solubility. Research approaches to overcome these challenges include optimizing expression systems (bacterial, yeast, insect, or mammalian cells), with mammalian expression systems often providing superior folding for mammalian transmembrane proteins. Fusion tags such as His6, GST, or MBP can enhance solubility, though their effect on protein activity must be carefully assessed. Detergent screening is critical for solubilizing membrane proteins, with mild non-ionic detergents like DDM or CHAPS often preserving protein structure. Proper refolding protocols may be necessary if the protein is expressed in inclusion bodies, and cryo-EM or X-ray crystallography can validate proper folding of the purified protein.

How does Mouse Tmco2 interact with protein complexes in neurodegenerative disease models?

Mouse Tmco2 likely forms specific protein interactions that may be relevant to neurodegenerative disease processes, particularly given the established interaction between human TMCC2 and amyloid protein precursor (APP). When investigating these interactions, researchers should employ co-immunoprecipitation, proximity ligation assays, and FRET/BRET approaches to identify binding partners in both healthy and disease model systems. Targeted gene editing using CRISPR/Cas9 to modify Tmco2 binding domains can elucidate the functional significance of specific interactions. In Alzheimer's disease models, special attention should be given to potential Tmco2 interactions with amyloid processing machinery, given that human TMCC2 (a related protein) associates with senile plaques and neuronal dystrophies in AD brain tissue .

What is the relationship between Tmco2 expression and neuroinflammatory pathways in mouse models?

The relationship between Tmco2 expression and neuroinflammatory pathways represents an emerging area of research with potential implications for neurodegenerative conditions. To investigate this relationship, researchers should examine Tmco2 expression in response to inflammatory stimuli using qPCR, western blotting, and in situ hybridization. Cell-type specific analyses using single-cell RNA sequencing can determine whether Tmco2 expression changes are specific to neurons, astrocytes, or microglia during neuroinflammation. Pathway analysis using RNA-seq data from inflammatory challenge models can position Tmco2 within specific signaling networks. Given that neuroinflammation is a key component of Alzheimer's disease, and human TMCC2 has been linked to AD pathology , understanding how mouse Tmco2 responds to inflammatory stimuli may provide valuable insights into conserved disease mechanisms.

What are the optimal conditions for expressing recombinant Mouse Tmco2 in bacterial systems?

Expressing recombinant mouse Tmco2 in bacterial systems requires careful optimization of several parameters. The following protocol synthesizes current best practices:

• Expression system selection: Use E. coli BL21(DE3) or Rosetta strains to accommodate potential rare codons in the Tmco2 sequence.

• Vector design: Clone the Tmco2 coding sequence into a pET vector system (such as pET-28a) with an N-terminal His-tag for purification purposes .

• Transformation protocol:

  • Transform expression plasmids into competent cells using heat-shock

  • Plate on selective media containing appropriate antibiotics

  • Confirm positive transformants by colony PCR or restriction digest

• Expression conditions:

  • Culture in LB media supplemented with appropriate antibiotics

  • Induce at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG

  • Lower induction temperature to 16-25°C to improve protein folding

  • Extend expression time to 16-20 hours for improved yield

• Harvest and lysis:

  • Harvest cells by centrifugation at 5000g for 15 minutes

  • Resuspend in buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, and protease inhibitors

  • Lyse cells by sonication or French press

• Purification strategy:

  • Purify using nickel-NTA affinity chromatography

  • Elute with imidazole gradient (50-500 mM)

  • Further purify by size exclusion chromatography if necessary

• Quality control:

  • Verify purity by SDS-PAGE

  • Confirm identity by western blot using anti-His antibodies and anti-Tmco2 specific antibodies

How can researchers effectively analyze Tmco2 interaction with APP-related proteins?

To analyze Tmco2 interactions with APP-related proteins, researchers should employ a multi-method approach:

• Co-immunoprecipitation (Co-IP):

  • Prepare cellular lysates under non-denaturing conditions

  • Immunoprecipitate Tmco2 using specific antibodies

  • Analyze co-precipitated proteins by western blotting for APP and related proteins

  • Perform reciprocal Co-IP to confirm interaction

• Proximity Ligation Assay (PLA):

  • Fix cells expressing both Tmco2 and potential binding partners

  • Use primary antibodies against each protein

  • Employ species-specific PLA probes

  • Quantify fluorescent signals indicating proximity (<40 nm)

• BiFC (Bimolecular Fluorescence Complementation):

  • Generate fusion constructs of Tmco2 and APP with split fluorescent protein fragments

  • Co-express in appropriate cell lines

  • Analyze reconstitution of fluorescence signal indicating interaction

  • Map interaction domains through truncation or mutation studies

• Pull-down assays with recombinant proteins:

  • Express and purify recombinant Tmco2 with affinity tags

  • Perform pull-down experiments with brain lysates

  • Analyze bound proteins by mass spectrometry and western blotting

  • Validate direct interactions with surface plasmon resonance

This comprehensive approach can reveal both direct and indirect interactions between Tmco2 and APP-related proteins, especially valuable given the established association between human TMCC2 and APP in Alzheimer's disease contexts .

What are the best methods for studying Tmco2 function in neuronal cell models?

Studying Tmco2 function in neuronal cell models requires integrating genetic, biochemical, and imaging approaches:

• Loss-of-function studies:

  • CRISPR/Cas9 gene editing to create Tmco2 knockout lines

  • siRNA or shRNA-mediated knockdown for transient reduction

  • Analyze phenotypes including morphology, synapse formation, and electrophysiology

• Gain-of-function approaches:

  • Overexpress wild-type or tagged Tmco2 constructs

  • Use inducible expression systems to control timing

  • Assess effects on neuronal differentiation and function

• Live-cell imaging:

  • Generate fluorescently tagged Tmco2 constructs

  • Perform time-lapse microscopy to track protein localization

  • Use photoactivatable or photoswitchable tags to monitor protein dynamics

• Subcellular localization analysis:

  • Perform subcellular fractionation to identify Tmco2-enriched compartments

  • Use immunocytochemistry with organelle markers to determine precise localization

  • Employ super-resolution microscopy for detailed spatial information

• Functional assays:

  • Calcium imaging to assess neuronal activity

  • Neurite outgrowth and axon guidance assays

  • Synaptogenesis and spine morphology analyses

  • Electrophysiological recordings to assess neuronal function

• Stress response studies:

  • Challenge neurons with oxidative stress, excitotoxicity, or protein misfolding stressors

  • Analyze Tmco2 expression, localization, and modification changes

  • Determine if Tmco2 manipulation affects neuronal survival under stress conditions

These methodologies provide a comprehensive assessment of Tmco2's roles in neuronal physiology and potentially pathophysiology.

What experimental controls are essential when studying Mouse Tmco2 in Alzheimer's disease models?

When studying mouse Tmco2 in Alzheimer's disease models, the following controls are essential to ensure robust, interpretable results:

• Genetic controls:

  • Include wild-type littermates for all transgenic models

  • Use isogenic cell lines for in vitro studies

  • Employ scrambled/non-targeting constructs for RNA interference experiments

  • Include empty vector controls for overexpression studies

• Age and sex-matched controls:

  • Use animals of the same age and sex distribution

  • Control for estrous cycle effects in female mice

  • Include longitudinal sampling points for age-dependent phenotypes

• Antibody validation controls:

  • Confirm antibody specificity using Tmco2 knockout tissues/cells

  • Include peptide competition assays

  • Test multiple antibodies targeting different epitopes

• Histological controls:

  • Use anatomically matched brain regions across samples

  • Include both affected and unaffected brain regions

  • Implement stereological sampling methods for quantification

• Model validation:

  • Verify amyloid pathology using established markers

  • Confirm cognitive/behavioral phenotypes characteristic of the model

  • Document expression levels of key AD-associated proteins

• Technical controls for protein interaction studies:

  • Include IgG controls for immunoprecipitation

  • Perform reverse co-immunoprecipitation

  • Test interactions under different detergent conditions

  • Use TMCC2 as a positive control given its established association with APP

• Pharmacological controls:

  • Include vehicle controls for all drug treatments

  • Test dose-dependency of effects

  • Implement appropriate washout periods

Proper implementation of these controls ensures that observed effects can be specifically attributed to Tmco2 rather than to experimental artifacts or model-specific peculiarities.

How should researchers interpret contradictory findings regarding Tmco2 expression in different mouse models?

When confronted with contradictory findings regarding Tmco2 expression across different mouse models, researchers should implement a systematic analytical approach:

• Model characterization comparative analysis:

  • Compare genetic backgrounds of mouse models (C57BL/6, BALB/c, etc.)

  • Assess age, sex, and environmental conditions across studies

  • Evaluate disease model induction methods (transgenic, chemical, infection)

• Methodological variation assessment:

  • Compare RNA quantification techniques (qPCR, RNA-seq, microarray)

  • Evaluate protein detection methods (western blot, ELISA, mass spectrometry)

  • Consider tissue preparation differences (fresh vs. fixed, extraction methods)

• Resolution approaches:

  • Conduct direct side-by-side comparisons using standardized protocols

  • Perform meta-analysis of expression data with statistical correction for model variables

  • Use multiple detection methods within the same experiment

• Biological context interpretation:

  • Consider Tmco2 expression in relation to neuroinflammatory markers

  • Evaluate expression in specific cell types rather than whole tissue

  • Assess temporal dynamics of expression changes

• Cross-validation strategies:

  • Compare findings with human data where available

  • Validate in vitro with primary cultures from multiple mouse strains

  • Test expression in response to standardized stimuli across models

This systematic approach helps distinguish genuine biological variation from technical artifacts, providing a foundation for reconciling apparently contradictory results.

What statistical approaches are most appropriate for analyzing Tmco2 expression changes in disease models?

When analyzing Tmco2 expression changes in disease models, researchers should select statistical approaches based on experimental design and data characteristics:

• For simple two-group comparisons:

  • Student's t-test for normally distributed data

  • Mann-Whitney U test for non-parametric data

  • Calculate effect sizes (Cohen's d) in addition to p-values

• For multi-group comparisons:

  • One-way ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • Kruskal-Wallis with Dunn's post-hoc for non-parametric data

  • Control for multiple comparisons using false discovery rate methods

• For longitudinal studies:

  • Repeated measures ANOVA or mixed-effects models

  • Area under the curve (AUC) analysis for time-course data

  • Time-series analysis for complex temporal patterns

• For correlation analyses:

  • Pearson or Spearman correlation between Tmco2 levels and pathological markers

  • Multiple regression to control for confounding variables

  • Path analysis for complex relationship networks

• For high-dimensional data:

  • Principal component analysis to identify major sources of variation

  • Hierarchical clustering to identify expression patterns

  • Machine learning approaches for complex pattern recognition

• For meta-analysis across studies:

  • Random-effects models to account for between-study heterogeneity

  • Forest plots to visualize effect sizes across studies

  • Funnel plots to assess publication bias

• Power analysis considerations:

  • Conduct a priori power analysis to determine sample size

  • Report effect sizes and confidence intervals

  • Consider biological significance alongside statistical significance

These approaches ensure robust analysis of Tmco2 expression data, facilitating meaningful biological interpretations and comparisons across studies.

How do findings from Mouse Tmco2 studies translate to human neurological disorders?

Translating findings from mouse Tmco2 studies to human neurological disorders requires careful consideration of several factors:

• Evolutionary conservation analysis:

  • Sequence homology between mouse Tmco2 and human TMCO2 (approximately 80-90% for most transmembrane proteins)

  • Comparison of protein domain structure and critical functional regions

  • Assessment of conservation of regulatory elements and splice variants

• Expression pattern comparison:

  • Evaluate tissue distribution in mouse versus human samples

  • Compare cell-type specific expression using single-cell RNA-seq data

  • Assess developmental expression trajectories across species

• Functional pathway conservation:

  • Determine conservation of binding partners and protein interactions

  • Compare involvement in signaling pathways between species

  • Evaluate phenotypic consequences of gene disruption

• Relevance to human pathology:

  • Analyze human brain tissue for TMCO2 abnormalities in neurodegenerative conditions

  • Correlate findings with those in mouse models of similar conditions

  • Consider the established association between the related protein TMCC2 and Alzheimer's disease pathology in humans

• Translational limitations:

  • Acknowledge species differences in brain architecture and complexity

  • Consider differences in lifespan and disease progression timelines

  • Recognize variations in immune responses between mice and humans

• Validation approaches:

  • Confirm key findings in human iPSC-derived neurons

  • Use post-mortem human brain tissue studies for validation

  • Correlate mouse findings with human genetic associations

This translational framework helps researchers interpret mouse Tmco2 findings in the context of human disease, particularly for neurological disorders where human TMCC2 has shown associations with pathology.

What are the most promising future research directions for Mouse Tmco2 studies?

The most promising future research directions for mouse Tmco2 studies span multiple dimensions of neurobiological research:

• Structural biology approaches:

  • Determine the high-resolution structure of Tmco2 using cryo-EM or X-ray crystallography

  • Map protein interaction domains precisely

  • Develop structure-based therapeutic modulators

• Systems biology integration:

  • Position Tmco2 within broader neuronal protein-protein interaction networks

  • Apply multi-omics approaches to understand Tmco2's role in cellular homeostasis

  • Develop computational models of Tmco2 function in neuronal systems

• Neurodevelopmental implications:

  • Characterize Tmco2's role in neuronal differentiation and circuit formation

  • Investigate potential contributions to neurodevelopmental disorders

  • Study temporal regulation of Tmco2 expression throughout brain development

• Therapeutic targeting potential:

  • Evaluate Tmco2 as a drug target or biomarker for neurological conditions

  • Develop antibodies or small molecules that modulate Tmco2 function

  • Assess viral vector approaches for Tmco2 gene therapy

• Comparative neurobiology:

  • Extend studies to non-human primates to better bridge mouse and human findings

  • Investigate evolutionary adaptations in Tmco2 structure and function

  • Compare roles across species with varying neurological complexity

• Advanced disease modeling:

  • Generate knock-in mouse models with human TMCO2 to better model human conditions

  • Develop organoid systems to study Tmco2 in complex cellular environments

  • Apply spatially resolved transcriptomics to map Tmco2 expression in intact brain circuits

These research directions will advance understanding of Tmco2's fundamental biology and potential relevance to human neurological disorders, building on the established connection between related proteins like TMCC2 and Alzheimer's disease pathology .