Recombinant Macaca fascicularis Integral membrane protein 2C (ITM2C)

What is ITM2C and what are its primary functions in biological systems?

ITM2C is a type II integral membrane protein primarily expressed in brain tissue. Its key functions include:

• Acting as a negative regulator of amyloid-beta peptide production by inhibiting the processing of amyloid precursor protein (APP)

• Blocking access of alpha- and beta-secretases to APP, thereby reducing amyloid-beta peptide generation

• Participating in immune modulation through interactions with beta-secretase (BACE1) and microtubule-destabilizing proteins

• Influencing neuronal differentiation and TNF-induced cell death

These functions position ITM2C as a potential therapeutic target for neurodegenerative conditions like Alzheimer's disease and as a biomarker in certain cancers.

Why use Macaca fascicularis (cynomolgus monkey) as a source for ITM2C in research?

Macaca fascicularis serves as an excellent model for human disease research because:

• Cynomolgus monkeys are phylogenetically close to humans, making their proteins highly homologous to human counterparts

• They are amenable to reproductive experimentation and provide valuable insights for translational research

• Their neurological systems closely mirror human systems, particularly important for studying brain-expressed proteins like ITM2C

• Using recombinant Macaca fascicularis ITM2C allows researchers to study a protein that closely resembles human ITM2C while avoiding ethical concerns of human tissue use

What expression systems are most effective for producing functional recombinant Macaca fascicularis ITM2C?

Several expression systems can be used, each with specific advantages:

For structural studies and applications not requiring post-translational modifications, E. coli-expressed ITM2C (as described in search result ) is often sufficient. For functional studies investigating protein-protein interactions, mammalian expression systems may provide more biologically relevant protein.

What are the optimal storage and handling conditions for maintaining recombinant ITM2C stability?

For maximum stability and activity retention:

• Store lyophilized protein at -20°C/-80°C for long-term storage

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Consider adding glycerol (final concentration 5-50%, with 50% being standard) for long-term storage

• After reconstitution, aliquot the protein to minimize freeze-thaw cycles

How can I verify the purity and functionality of recombinant ITM2C?

Multiple analytical approaches should be employed:

• Purity assessment:

  • SDS-PAGE analysis (expect >90% purity for high-quality preparations)

  • Western blot using anti-ITM2C antibodies

  • Mass spectrometry for precise molecular weight determination

• Functional verification:

  • Binding assays with known interaction partners (APP, secretases)

  • Activity assays measuring inhibition of amyloid-beta production

  • Circular dichroism to confirm proper protein folding

• Application-specific validation:

  • For Alzheimer's studies: APP processing inhibition assays

  • For cancer research: Expression analysis relative to control samples

How does ITM2C regulate amyloid-beta peptide production and what experimental approaches best demonstrate this?

ITM2C regulates amyloid-beta production through direct inhibition of APP processing:

• It blocks access of both alpha- and beta-secretases to APP, thereby preventing cleavage that would generate amyloid-beta peptides

• This mechanism positions ITM2C as a potential therapeutic target for Alzheimer's disease

Experimental approaches to demonstrate this regulatory function include:

• Co-immunoprecipitation studies:

  • Precipitate ITM2C and probe for APP and secretases to confirm physical interaction

  • Compare binding affinities of wild-type versus mutant ITM2C

• Cell-based assays:

  • Overexpress or knockdown ITM2C in neuronal cell lines

  • Measure changes in amyloid-beta levels using ELISA

  • Visualize subcellular localization of ITM2C and APP using fluorescence microscopy

• In vitro enzyme inhibition assays:

  • Incubate purified secretases with fluorogenic APP-derived substrates

  • Add increasing concentrations of recombinant ITM2C

  • Measure changes in enzyme activity through fluorescence detection

What are the methodological challenges in studying ITM2C in the context of neurodegeneration?

Key challenges include:

• Protein-protein interaction complexity:

  • ITM2C interacts with multiple partners in dynamic complexes

  • Solution: Use proximity labeling techniques (BioID, APEX) to capture transient interactions

• Membrane protein solubility:

  • As a membrane protein, ITM2C requires careful solubilization

  • Solution: Optimize detergent conditions (try CHAPS, DDM, or digitonin) or use nanodiscs

• Translating findings between species:

  • Despite homology, species differences exist between monkey and human ITM2C

  • Solution: Perform comparative studies with human ITM2C when possible; use bioinformatic analysis to predict functional conservation

• Temporal dynamics of APP processing:

  • APP processing is regulated by multiple factors over time

  • Solution: Develop time-course experiments and real-time imaging of processing events

How can recombinant ITM2C be incorporated into drug discovery for Alzheimer's disease?

Recombinant ITM2C can facilitate drug discovery through:

• High-throughput screening platforms:

  • Develop assays measuring ITM2C-APP or ITM2C-secretase interactions

  • Screen compound libraries for molecules that enhance ITM2C's inhibitory activity

  • Use SPR or FRET-based approaches for interaction studies

• Structure-based drug design:

  • Determine the 3D structure of ITM2C (or key domains) via X-ray crystallography or cryo-EM

  • Identify binding pockets that could be targeted by small molecules

  • Design compounds that stabilize ITM2C-APP interaction or enhance secretase inhibition

• Functional validation:

  • Test candidate compounds in neuronal cell lines expressing ITM2C

  • Measure effects on amyloid-beta production

  • Validate in animal models of Alzheimer's disease

What evidence supports ITM2C as a biomarker in multiple myeloma and colorectal cancer?

The evidence for ITM2C as a cancer biomarker includes:

This evidence suggests ITM2C may serve both as a prognostic marker and potentially as part of diagnostic gene signatures.

What experimental approaches are recommended for studying ITM2C's role in cancer progression?

To investigate ITM2C in cancer:

• Expression analysis:

  • Quantify ITM2C expression in tumor vs. normal tissues using qPCR, western blot, and immunohistochemistry

  • Correlate expression with clinical outcomes using Kaplan-Meier analysis

  • Perform single-cell RNA sequencing to identify cell populations expressing ITM2C

• Functional studies:

  • Generate cancer cell lines with ITM2C knockdown or overexpression

  • Assess effects on proliferation, migration, invasion, and apoptosis

  • Evaluate changes in known cancer signaling pathways (WNT, MAPK, etc.)

• Mechanistic investigations:

  • Identify ITM2C-interacting proteins in cancer cells using co-IP followed by mass spectrometry

  • Determine if ITM2C's role in APP processing relates to its cancer functions

  • Investigate potential immunomodulatory functions in the tumor microenvironment

How can the correlation between ITM2C, CA2, and CA7 be leveraged in colorectal cancer research?

This gene correlation can be utilized through:

• Biomarker development:

  • Design multiplexed qPCR assays targeting all three genes

  • Develop immunohistochemistry panels for simultaneous detection

  • Create machine learning algorithms integrating expression data

• Mechanistic studies:

  • Investigate potential functional relationships between these proteins

  • Determine if they participate in common pathways or protein complexes

  • Study the impact of modulating all three genes simultaneously

• Therapeutic targeting:

  • Explore whether targeting these genes in combination provides synergistic effects

  • Develop therapeutic approaches that normalize the expression of all three genes

  • Screen for compounds that specifically affect this gene signature

The strong correlation (CA2-ITM2C: 0.79-0.88; CA7-ITM2C: 0.72-0.73) suggests these genes may be co-regulated or functionally related, providing a foundation for deeper mechanistic studies.

What detection systems and antibodies are validated for studies involving recombinant Macaca fascicularis ITM2C?

Validated detection systems include:

• Western blot:

  • Rabbit polyclonal antibodies against ITM2C have been validated for detecting recombinant ITM2C in human, mouse, and rat samples

  • Recommended dilution ranges: 1:500-1:2000 for primary antibodies

  • Use His-tag antibodies for detection of recombinant His-tagged ITM2C

• ELISA:

  • Commercial sandwich ELISA kits with verified cross-reactivity to Macaca fascicularis ITM2C

  • Develop custom ELISA using purified recombinant ITM2C as a standard

• Immunocytochemistry/Immunohistochemistry:

  • Optimize fixation conditions (4% paraformaldehyde recommended)

  • Use antigen retrieval for tissue sections (citrate buffer, pH 6.0)

  • Validate antibody specificity using ITM2C-knockout controls

How can researchers design experiments to investigate ITM2C interactions with APP and secretases?

Effective experimental designs include:

• Proximity-based interaction studies:

  • Bimolecular Fluorescence Complementation (BiFC): Tag ITM2C and potential partners with complementary fluorescent protein fragments

  • FRET/BRET: Use fluorescent/bioluminescent tags to measure real-time interactions

  • Proximity Ligation Assay (PLA): Visualize interactions at endogenous expression levels

• Domain mapping experiments:

  • Create truncation mutants of ITM2C to identify interaction domains

  • Use peptide arrays to pinpoint specific binding motifs

  • Perform alanine scanning mutagenesis of key residues

• Membrane-specific techniques:

  • Utilize supported lipid bilayers reconstituted with purified proteins

  • Apply crosslinking approaches optimized for membrane environments

  • Employ super-resolution microscopy to visualize clustering in membranes

What computational approaches can enhance ITM2C research and provide new insights?

Advanced computational methods include:

• Structural prediction and analysis:

  • AlphaFold2 or RoseTTAFold for predicting ITM2C structure

  • Molecular dynamics simulations to study conformational changes

  • Docking studies to predict interactions with APP, secretases, or potential therapeutics

• Systems biology approaches:

  • Network analysis to place ITM2C in broader cellular pathways

  • Multi-omics integration combining proteomics, transcriptomics, and metabolomics data

  • Machine learning to identify patterns in experimental data and predict outcomes

• Bioinformatic analysis across species:

  • Comparative genomics between human, Macaca fascicularis, and other species

  • Evolutionary analysis to identify conserved functional domains

  • Single-cell transcriptomic analysis to map cell type-specific expression patterns