Recombinant TM2 domain-containing protein C02F5.13 (C02F5.13)

What is the structural composition of TM2 domain-containing protein C02F5.13?

TM2 domain-containing protein C02F5.13 is a precursor protein from Caenorhabditis elegans with the UniProt accession number P61228 (UniProt ID: TM2D1_CAEEL). Like other TM2D family proteins, it possesses a predicted N-terminal signal sequence and two transmembrane domains connected by a short intracellular loop. This loop contains an evolutionarily conserved DRF (aspartate-arginine-phenylalanine) motif, which is found in some G-protein coupled receptors and mediates conformational changes upon ligand binding. The protein has extracellular regions between the signal sequence and first transmembrane domain that show divergence across species, while the transmembrane domains and intracellular loop sequence are highly conserved. The C-terminal extracellular tail is also evolutionarily conserved but varies among the three TM2D protein types .

How is C02F5.13 classified within protein databases?

C02F5.13 is classified in the UniProt database with the accession number P61228. It is designated as a "TM2 domain-containing protein C02F5.13 precursor" with the UniProt ID "TM2D1_CAEEL." In the Protein Ontology (PRO) database, it has the PRO ID "PR:P61228" with the PRO name "TM2 domain-containing protein C02F5.13 (worm)" and the short label "TM2D2 (worm)." It falls under the "organism-gene" category in the PRO classification system .

What is known about the evolutionary conservation of TM2D proteins?

TM2D proteins are conserved across metazoans and are encoded by three separate genes in each species (TM2D1, TM2D2, and TM2D3). Each protein is encoded by a highly conserved orthologous gene. In Drosophila, these genes are CG10795 (corresponding to TM2D1), CG11103 (corresponding to TM2D2), and amx (corresponding to TM2D3). The sequences of the two transmembrane domains and the intracellular loop containing the DRF motif are highly conserved throughout evolution. In contrast, the extracellular regions between the signal sequence and the first transmembrane domain show divergence across species and among the three TM2D proteins .

What are the recommended approaches for recombinant expression of C02F5.13?

Methodological Answer:

When designing experiments for recombinant expression of C02F5.13, researchers should consider the following approach:

• Vector Selection: Choose expression vectors compatible with the host system (bacterial, yeast, insect, or mammalian). For membrane proteins like C02F5.13, eukaryotic expression systems (particularly insect or mammalian cells) are often preferred to ensure proper folding and post-translational modifications.

• Affinity Tag Selection: Incorporate appropriate affinity tags (His, GST, FLAG) at either the N- or C-terminus, being mindful that the N-terminus contains a signal sequence that may be cleaved during processing.

• Codon Optimization: Optimize codons for the expression host to enhance protein production.

• Solubilization Strategy: Develop effective membrane extraction protocols using mild detergents like DDM or CHAPS that preserve protein structure.

• Purification Protocol: Implement a two-step purification process combining affinity chromatography and size exclusion chromatography to obtain pure protein.

• Quality Control: Verify protein identity and integrity using mass spectrometry, circular dichroism, and thermal shift assays to confirm proper folding .

How should functional assays be designed to study C02F5.13's role in Notch signaling?

Methodological Answer:

Based on studies of TM2D family proteins, functional assays should be designed with several key considerations:

• Genetic Approaches: CRISPR/Cas9-mediated gene editing can be used to generate knockout or knock-in C. elegans models to assess phenotypic effects. Previous studies with TM2D gene knockouts in Drosophila revealed maternal-effect neurogenic phenotypes that should be specifically examined.

• Epistasis Experiments: To determine where in the Notch signaling pathway C02F5.13 functions, design epistatic analyses using combinations of established Notch pathway mutations and C02F5.13 mutations.

• Protein-Protein Interaction Assays: Implement co-immunoprecipitation, proximity ligation assays, or yeast two-hybrid screens to identify potential interactions with Notch pathway components, particularly γ-secretase complex components.

• Reporter Assays: Utilize Notch-responsive luciferase or GFP reporters to quantitatively measure the effects of C02F5.13 manipulation on Notch signaling output.

• Domain Function Analysis: Create truncated versions of C02F5.13 (similar to the Amx ΔECD construct used in Drosophila studies) to assess the functional importance of specific protein regions. Previous research showed that truncated forms containing only the highly conserved regions acted as potent inhibitors of Notch signaling when overexpressed .

What experimental approaches are appropriate for investigating protein-protein interactions involving C02F5.13?

Methodological Answer:

To rigorously investigate protein-protein interactions involving C02F5.13, a multi-faceted experimental approach is recommended:

• Yeast Two-Hybrid (Y2H) Screening: While traditional Y2H may be challenging for membrane proteins, modified membrane Y2H systems or split-ubiquitin Y2H approaches can be employed. This approach was successfully used to identify TM2D1's interaction with Aβ peptides.

• Co-Immunoprecipitation (Co-IP): Use tagged versions of C02F5.13 to pull down interaction partners from cellular lysates, followed by mass spectrometry identification of binding partners.

• Bimolecular Fluorescence Complementation (BiFC): Fuse potential interaction partners with complementary fragments of a fluorescent protein to visualize interactions in living cells.

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics, recombinant C02F5.13 can be immobilized on a sensor chip and potential binding partners flowed over to measure association and dissociation constants.

• Proximity-Based Labeling: Techniques such as BioID or APEX2 can identify proteins that come into close proximity with C02F5.13 in living cells, even if interactions are transient.

• Cross-Linking Mass Spectrometry: Chemical cross-linking followed by mass spectrometry can capture and identify interaction interfaces between C02F5.13 and binding partners .

How does C02F5.13 potentially modulate γ-secretase activity in the context of Notch signaling?

Methodological Answer:

Investigating C02F5.13's modulation of γ-secretase requires sophisticated experimental approaches:

• In Vitro γ-Secretase Activity Assays: Reconstitute purified γ-secretase components with and without recombinant C02F5.13 to measure effects on enzymatic activity using fluorogenic substrates.

• Cell-Based Notch Cleavage Assays: Utilize reporter constructs that distinguish between different Notch cleavage events (particularly S3 cleavage) to determine specific effects of C02F5.13 manipulation.

• Genetic Interaction Studies: Create double mutants of C02F5.13 and γ-secretase components (particularly presenilin homologs) to assess phenotypic enhancement or suppression in C. elegans.

• Structural Analysis: Implement cryo-EM studies of γ-secretase complexes with and without C02F5.13 to determine potential binding sites and conformational changes.

• Domain-Specific Effects: Based on findings with Drosophila Amx ΔECD, conduct parallel experiments with truncated versions of C02F5.13 to identify regions critical for γ-secretase interaction.

Research in Drosophila demonstrated that overexpression of a truncated form of Amx (TM2D3 homolog) inhibited Notch signaling at the S3 cleavage step, which is mediated by γ-secretase. Similar approaches comparing full-length versus truncated C02F5.13 could reveal conserved mechanisms .

What is the relationship between TM2D family proteins and neurodegenerative diseases?

Methodological Answer:

Investigating the relationship between C02F5.13 and neurodegenerative diseases requires a comprehensive research approach:

• Comparative Analysis: Compare C02F5.13 with its human homologs, particularly focusing on the conserved domains that have been implicated in Alzheimer's disease (AD) pathways.

• Amyloid-β Interaction Studies: Based on studies showing TM2D1 can bind Aβ42 and Aβ40 peptides, conduct binding assays with recombinant C02F5.13 and Aβ peptides to determine if this function is conserved.

• Lifespan and Neurodegeneration Analysis: Assess whether C02F5.13 mutants in C. elegans display altered lifespan or progressive neurodegeneration phenotypes similar to those observed with TM2D3/Amx in Drosophila.

• Cellular Toxicity Models: Develop cellular models expressing C02F5.13 and expose them to aggregated Aβ to test whether C02F5.13 affects cellular vulnerability, similar to studies with TM2D1 in human neuroblastoma cells.

• DRF Motif Mutagenesis: Since the DRF motif in TM2D1 was required for mediating Aβ-toxicity, create point mutations in the corresponding motif of C02F5.13 to test functional conservation.

How do the functions of C02F5.13 compare with other TM2D family members in different species?

Methodological Answer:

A comprehensive comparative analysis of TM2D family proteins requires several methodological approaches:

• Multiple Sequence Alignment: Conduct detailed sequence alignments of all three TM2D proteins across multiple species, focusing particularly on the conserved transmembrane domains and intracellular loop regions.

• Heterologous Complementation Experiments: Test whether C02F5.13 can functionally substitute for TM2D homologs in other species by expressing it in knockout models of Drosophila or mammalian cells.

• Parallel Phenotypic Analysis: Generate and characterize C. elegans strains with mutations in all TM2D homologs, individually and in combination, similar to the studies in Drosophila that showed single, double, and triple knockouts shared similar maternal-effect neurogenic phenotypes.

• Domain Swap Experiments: Create chimeric proteins that combine domains from different TM2D family members to identify which regions confer specific functional properties.

• Comparative Expression Profiling: Analyze and compare the expression patterns of all TM2D genes across tissues and developmental stages in C. elegans and other model organisms.

Research in Drosophila revealed that knockouts of TM2D1, TM2D2, and TM2D3 homologs showed indistinguishable maternal-effect neurogenic phenotypes, suggesting functional redundancy. Triple knockout animals displayed the same phenotype as single mutants, indicating these genes function together in specific contexts .

How should researchers address conflicting data regarding C02F5.13's molecular function?

Methodological Answer:

When faced with conflicting results regarding C02F5.13's function, researchers should implement a systematic approach to data reconciliation:

• Context-Dependent Analysis: Evaluate whether discrepancies arise from differences in experimental conditions, cell types, or model organisms. For instance, results from yeast systems may differ from those in C. elegans due to differences in the cellular machinery.

• Technical Validation: Confirm key findings using complementary techniques. If protein-protein interactions were identified via yeast two-hybrid, validate them using co-immunoprecipitation or proximity ligation assays.

• Dose-Dependency Assessment: Determine whether conflicting observations result from concentration-dependent effects by conducting detailed dose-response experiments.

• Temporal Analysis: Examine whether conflicting functions occur at different developmental stages or under different cellular conditions.

• Meta-Analysis Framework: When literature presents contradictory findings, implement a formal meta-analysis methodology to identify patterns across studies and potential sources of heterogeneity.

This approach would be particularly valuable when reconciling conflicting data such as that observed with TM2D1, where one study suggested it functions as a receptor mediating Aβ-toxicity, while a follow-up study indicated it is not coupled to G proteins as originally hypothesized .

What statistical approaches are most appropriate for analyzing TM2D protein interaction data?

Methodological Answer:

Analyzing protein interaction data for TM2D proteins requires specialized statistical approaches:

• Multiple Testing Correction: When screening for multiple potential interaction partners, implement Bonferroni or Benjamini-Hochberg corrections to control for false discovery rates.

• Bayesian Network Analysis: For integrating diverse interaction datasets (Y2H, Co-IP, proximity labeling), Bayesian networks can identify high-confidence interactions supported by multiple lines of evidence.

• Statistical Significance Thresholds: For interaction scores like those presented in STRING database analyses, set appropriate confidence thresholds (typically scores >0.7 indicate high confidence).

• Enrichment Analysis: When identifying multiple interaction partners, conduct Gene Ontology or pathway enrichment analyses to identify functionally related groups.

• Control Selection: Implement proper statistical controls for background binding, including both positive controls (known interactors) and negative controls (proteins unlikely to interact).

The STRING database presents interaction scores for protein partners, with scores ranging from 0 to 1, where higher scores indicate greater confidence. For instance, C02F5.3 (a GTP-binding protein in C. elegans) shows interaction scores of 0.995 with T26E3.4 and 0.958 with F27D4.4, indicating high-confidence interactions .

What experimental designs are most appropriate for investigating the maternal-effect phenotypes associated with TM2D proteins?

Methodological Answer:

The maternal-effect neurogenic phenotypes observed with TM2D family proteins require specific experimental designs for thorough characterization:

• Genetic Mosaics: Generate genetic mosaics where the maternal germline is either wild-type or mutant for C02F5.13 to distinguish between maternal contribution and zygotic requirements.

• Temporal Rescue Experiments: Implement temporally controlled expression systems (heat-shock promoters or drug-inducible systems) to express C02F5.13 at specific developmental stages and determine critical periods.

• Single-Cell Transcriptomics: Apply single-cell RNA sequencing to embryos from wild-type and C02F5.13 mutant mothers to identify transcriptional changes that contribute to neurogenic phenotypes.

• Lineage Tracing: Combine C02F5.13 mutations with lineage markers to track specific cell fates and determine precisely which developmental decisions are altered.

• Pre-Experimental Research Design Planning: Implement true experimental research design principles by formulating clear hypotheses about C02F5.13's maternal function, identifying appropriate independent variables (genetic manipulations) and dependent variables (phenotypic outcomes) .

• Factorial Experimental Design: When testing interactions between multiple TM2D genes, use factorial designs that can detect both main effects and interaction effects between different gene manipulations .

How can researchers effectively compare recombinant TM2D proteins from different species?

Methodological Answer:

Comparing recombinant TM2D proteins across species requires systematic analytical approaches:

• Standardized Expression Systems: Express all TM2D variants (human, mouse, Drosophila, C. elegans) in the same host system to minimize system-specific variations in post-translational modifications.

• Parallel Purification Protocols: Implement identical purification schemes for all proteins, with careful calibration of detergent conditions for membrane protein extraction.

• Comparative Structural Analysis: Utilize circular dichroism spectroscopy and thermal shift assays to compare secondary structure composition and stability across species variants.

• Functional Equivalence Testing: Develop cross-species complementation assays where TM2D proteins from one species are tested for functional rescue in another species.

• Domain-Specific Conservation Analysis: Create a comparative matrix of functional properties associated with specific domains, with particular focus on the highly conserved transmembrane domains and intracellular loop.

These approaches would build on the established conservation patterns where transmembrane domains and intracellular loops of TM2D proteins show high conservation across species, while extracellular regions display greater divergence .

What methodological approaches should be used when investigating potential redundancy between TM2D family members?

Methodological Answer:

Investigating functional redundancy among TM2D family members requires comprehensive experimental strategies:

• Combinatorial Genetic Analysis: Generate single, double, and triple knockout/knockdown models as was done in Drosophila, where single TM2D gene knockouts showed phenotypes indistinguishable from the triple knockout.

• Biochemical Redundancy Testing: Analyze whether different TM2D proteins can bind to the same partners by conducting competitive binding assays with purified components.

• Phenotypic Rescue Experiments: Test whether overexpression of one family member can rescue phenotypes caused by loss of another family member.

• Spatiotemporal Expression Analysis: Compare expression patterns of all TM2D genes using techniques like RNA in situ hybridization, reporter constructs, or single-cell transcriptomics to identify potential sites of redundancy.

• Dose-Dependency Studies: Investigate whether partial reduction of multiple TM2D genes produces synergistic effects that would indicate partial redundancy.

Research in Drosophila demonstrated that single knockouts of TM2D1, TM2D2, and TM2D3 homologs showed identical maternal-effect neurogenic phenotypes, and the triple knockout displayed the same phenotype, suggesting these genes function together rather than having distinct individual functions .

What multiperspectival approaches can enhance research on TM2D protein family function?

Methodological Answer:

Implementing multiperspectival approaches can provide more comprehensive insights into TM2D protein functions:

• Interdisciplinary Integration: Combine methodologies from structural biology, cell biology, genetics, and computational biology to develop a holistic understanding of TM2D protein function.

• Multi-Scale Analysis: Investigate TM2D proteins at multiple biological scales, from atomic structure to cellular function to organismal phenotypes, creating an integrated model spanning these scales.

• Methodological Triangulation: Use complementary methods to address the same research question, such as combining genetic, biochemical, and imaging approaches to validate key findings about C02F5.13 function.

• Comparative Systems Analysis: Study C02F5.13 simultaneously in multiple model systems (yeast, C. elegans, mammalian cells) to distinguish conserved from context-specific functions.

• Multi-Perspective Data Interpretation: Analyze results through different theoretical frameworks, such as developmental biology, evolutionary biology, and molecular medicine perspectives .

The multiperspectival approach integrates diverse research methodologies to create a more complete understanding of complex biological systems, allowing researchers to overcome the limitations inherent in any single approach .

How can researchers design less frequently used methodologies to study C02F5.13 functions?

Methodological Answer:

Researchers can implement innovative or less conventional methodologies to gain unique insights into C02F5.13 functions:

• Multimodal Analysis: Combine protein localization data with interaction data to create spatially resolved interaction networks, providing context for C02F5.13 function.

• Conversation Analysis Applied to Scientific Discourse: Analyze how scientific understanding of TM2D proteins has evolved through careful examination of the language and framing used in the literature over time.

• Grounded Theory Applications: Use inductive approaches to develop new theoretical frameworks for understanding membrane protein function based on empirical observations of C02F5.13 rather than testing pre-existing hypotheses.

• Phenomenological Approaches: Explore how C02F5.13's functions emerge from complex interactions within cellular systems rather than reducing it to individual molecular interactions.

• Hybrid Methodology Design: Develop custom methodological approaches that combine elements from established techniques, such as integrating CRISPR screening with mass spectrometry to simultaneously perturb and measure protein interaction networks involving C02F5.13 .

These approaches represent less frequently used research methodologies that might yield novel insights beyond what conventional approaches can provide, particularly for proteins with complex functions like TM2D family members .