Recombinant Rat Integral membrane protein 2C (Itm2c)

What is Integral Membrane Protein 2C (Itm2c) and what are its key structural features?

Integral Membrane Protein 2C (Itm2c) is a member of the BRI3 family of proteins. It is a type II transmembrane glycoprotein consisting of 267-269 amino acids in humans and rats, respectively. The protein contains a BRICHOS domain (approximately located at amino acids 136-230 in the human ortholog) which is thought to have a chaperone function . Rat Itm2c has a full-length sequence of 269 amino acids with a molecular weight of approximately 36-38 kDa .

The key structural features of Itm2c include:

• A short N-terminal cytoplasmic domain

• A single transmembrane domain

• A larger C-terminal extracellular domain containing the BRICHOS domain

• Potential proteolytic cleavage site (analogous to the human ortholog's furin cleavage site at Arg242-Gly243)

• Potential glycosylation sites that contribute to the final molecular weight

The complete amino acid sequence of rat Itm2c is: MVKISFQPAVAGVKAEKADKAAASGPASASAPAAEILLTPAREERPPRHRSRKGGSVGGVCYLSMGMVVLLMGLVFASVYIYRYFFLAQLARDNFFHCGVLYEDSLSSQIRTRLELEEDVKIYLEENYERINVPVPQFGGGDPADIIHDFQRGLTAYHDISLDKCYVIELNTTIVLPPRNFWELLMNVKRGTYLPQTYIIQEEMVVTEHVRDKEALGSFIYHLCNGKDTYRLRRRATRRRINKRGAKNCNAIRHFENTFVVETLICGVV .

What expression systems are commonly used for recombinant Itm2c production?

For recombinant rat Itm2c production, Escherichia coli (E. coli) is the most commonly documented expression system. This bacterial expression system offers several advantages for producing research-grade recombinant proteins:

• High protein yield

• Cost-effectiveness

• Scalability

• Well-established protocols

According to the product information, recombinant full-length rat Itm2c protein is typically expressed in E. coli with an N-terminal His tag . The expression construct generally contains the complete coding sequence (amino acids 1-269) of rat Itm2c fused to the tag.

Alternative expression systems that might be considered for specific research applications include:

• Mammalian expression systems (e.g., HEK293, CHO cells) - better for studying post-translational modifications

• Insect cell systems (e.g., Sf9, High Five) - compromise between bacterial and mammalian systems

• Yeast systems (e.g., Pichia pastoris) - for higher eukaryotic processing with better yields

The choice of expression system should be guided by the specific experimental requirements, particularly regarding protein folding, post-translational modifications, and functional activity needs.

What is the BRICHOS domain in Itm2c and what function does it serve?

The BRICHOS domain is a conserved structural motif found in several protein families, including Itm2c. In human ITM2C, the BRICHOS domain spans approximately amino acids 136-230 . This domain is named after three protein families in which it was first identified: BRI2 (ITM2B), Chondromodulin-I, and Surfactant protein C.

Key functions of the BRICHOS domain in Itm2c include:

• Chaperone activity: The domain is thought to have chaperone functions that help prevent protein misfolding and aggregation. This is particularly relevant for amyloidogenic proteins.

• Protein processing regulation: Evidence suggests the BRICHOS domain may regulate proteolytic processing, both of the Itm2c protein itself and its interacting partners.

• Protein-protein interactions: The domain may mediate interactions with other proteins, such as the documented interaction with amyloid precursor protein (APP) .

• Anti-amyloidogenic properties: Research on BRICHOS domains in related proteins suggests they may inhibit the formation of amyloid fibrils, which could be relevant to neurodegenerative disease research.

The BRICHOS domain structure consists of five α-helices and likely plays a crucial role in the biological functions of Itm2c, particularly in its potential neuroprotective mechanisms and interactions with proteins involved in neurodegeneration pathways.

How is recombinant rat Itm2c typically purified for research applications?

Purification of recombinant rat Itm2c typically involves a multi-step process that exploits the properties of both the protein and its affinity tags. Based on standard protocols for His-tagged proteins and the available product information, the typical purification workflow includes:

• Affinity chromatography: The primary purification step uses immobilized metal affinity chromatography (IMAC) with Ni-NTA or similar matrices to capture the His-tagged Itm2c protein . This step provides good initial purity.

• Buffer exchange/desalting: Following elution from the affinity column, the protein solution undergoes buffer exchange to remove imidazole and adjust salt concentration.

• Secondary chromatography: Depending on the required purity, additional chromatographic steps may include:

  • Ion exchange chromatography (based on charge properties)

  • Size exclusion chromatography (to separate any aggregates or truncated forms)

• Quality control: The purified protein undergoes verification by:

  • SDS-PAGE analysis to confirm purity (typically >90% for research applications)

  • Western blotting to confirm identity

  • Mass spectrometry for precise molecular weight determination

• Final formulation: The purified protein is typically formulated in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 for stability . The final product is often lyophilized for long-term storage.

For researchers working with recombinant rat Itm2c, it's important to note that proper reconstitution involves adding deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, with a recommendation to add 5-50% glycerol for long-term storage at -20°C/-80°C .

What is the amino acid sequence homology between rat Itm2c and human/mouse orthologs?

The Integral Membrane Protein 2C (Itm2c/ITM2C) is highly conserved across species, reflecting its important biological functions. Comparative analysis of the amino acid sequences reveals significant homology between rat, human, and mouse orthologs:

Sequence homology comparison:

*Estimated values based on the general conservation pattern reported for this protein family.

The high degree of sequence conservation, particularly in the extracellular domain containing the BRICHOS domain, suggests that:

• The protein's functional domains are evolutionarily conserved

• Studies using rat Itm2c may have translational relevance to human biology

• Critical functional residues and motifs are likely preserved across species

This homology information is valuable for researchers designing experiments that aim to:

• Extrapolate findings from rat models to human disease contexts

• Develop cross-species reactive tools (antibodies, inhibitors)

• Identify functionally important conserved residues for mutagenesis studies

The high sequence identity also suggests that the biological interactions of Itm2c with partners like APP and its role in processes such as protein processing may be conserved between these mammalian species.

What are the methodological considerations for studying Itm2c interactions with APP and SCG10?

When investigating Itm2c interactions with Amyloid Precursor Protein (APP) and Superior Cervical Ganglion 10 (SCG10), researchers should consider the following methodological approaches and technical considerations:

Experimental methods for detecting protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-Itm2c antibodies to pull down protein complexes and blot for APP or SCG10

  • Validate with reverse Co-IP using APP or SCG10 antibodies

  • Include appropriate negative controls (IgG, irrelevant antibodies)

• Proximity Ligation Assay (PLA):

  • Enables visualization of protein interactions in situ with single-molecule sensitivity

  • Particularly useful for membrane protein interactions like Itm2c-APP

• FRET/BRET analysis:

  • Tag proteins with appropriate fluorophore pairs for energy transfer

  • Allows real-time monitoring of interactions in living cells

Functional analysis of interactions:

• For APP processing studies:

  • Measure APP proteolytic fragments (Aβ, sAPPα, CTFs) by Western blot or ELISA

  • Compare processing in the presence/absence of Itm2c

  • Consider both overexpression and knockdown approaches

• For SCG10 microtubule studies:

  • Microtubule stability assays with purified proteins

  • Neurite outgrowth assays in primary neurons

  • Live-cell imaging of microtubule dynamics

Domain mapping considerations:

• Create truncated versions of Itm2c to identify interaction domains

• Pay special attention to the BRICHOS domain (aa 136-230 in human)

• Consider potential effects of post-translational modifications on interactions

Cell models for interaction studies:

• Neuronal cell lines (for physiological relevance)

• Primary neurons (for most physiological context)

• HEK293 cells (for high transfection efficiency in initial screening)

These methodological approaches should be adapted based on specific research questions and available resources. The membrane-bound nature of Itm2c presents particular challenges that may require specialized techniques for studying protein-protein interactions.

How can researchers effectively validate the functional activity of recombinant Itm2c?

Validating the functional activity of recombinant rat Itm2c requires a multi-faceted approach that addresses both its structural integrity and biological functions. Here are comprehensive strategies for functional validation:

Structural and biochemical validation:

• Proper folding assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Limited proteolysis to examine accessibility of cleavage sites

  • Size exclusion chromatography to detect proper oligomeric state (including potential dimerization)

• Post-translational modification analysis:

  • Mass spectrometry to identify any modifications

  • Glycosylation analysis using glycosidases and lectin binding

Functional validation approaches:

• APP processing inhibition assay:

  • Co-express recombinant Itm2c with APP in cell lines

  • Measure APP cleavage products by Western blot

  • Compare with native Itm2c and negative controls

  • Expected outcome: Itm2c binding should block proteolytic processing of APP

• SCG10 interaction assay:

  • In vitro binding assays with purified SCG10

  • Microtubule destabilization assays

  • Neurite outgrowth assays in neuronal cells

  • Expected outcome: Functional Itm2c should inhibit the microtubule-destabilizing activity of SCG10

• Autophagy regulation assessment:

  • Based on findings with the related protein ITM2A , examine:

    • LC3-II conversion by Western blot

    • p62 degradation kinetics

    • Autophagic vesicle formation by electron microscopy

    • mTOR pathway analysis through phospho-4EBP1 levels

Validation experimental design table:

These validation approaches should be selected based on the specific research questions and the intended use of the recombinant protein. Comparing results with those obtained using endogenous Itm2c from rat tissue samples provides an important reference point for functional validation.

What are the technical challenges in studying post-translational modifications of Itm2c?

Studying post-translational modifications (PTMs) of Itm2c presents several technical challenges that researchers should be aware of and plan for in their experimental design. Based on what we know about the Itm2c protein family, here are the key challenges and recommended approaches:

Challenges in Itm2c PTM analysis:

• Membrane protein nature:

  • Hydrophobicity complicates extraction and purification

  • Detergent requirements may interfere with some analytical methods

  • Retention of native conformation during extraction is difficult

• Low abundance of physiological PTMs:

  • Many PTMs occur on only a fraction of the total protein pool

  • Detection requires highly sensitive methods

  • Background signal can mask low-abundance modifications

• Heterogeneous glycosylation:

  • As a glycoprotein, Itm2c likely has complex glycan structures

  • Glycan heterogeneity complicates mass analysis

  • Functional significance of specific glycoforms is difficult to establish

• Phosphorylation detection:

  • Based on data from the related protein ITM2A , phosphorylation may be dynamic

  • Stimulus-dependent phosphorylation requires precise timing for detection

  • Site-specific phosphorylation antibodies may not be commercially available

Recommended methodological approaches:

• For glycosylation analysis:

  • Enzymatic deglycosylation followed by mobility shift analysis

  • Lectin affinity chromatography to enrich specific glycoforms

  • Mass spectrometry with glycopeptide enrichment

  • Glycan profiling using HILIC-UPLC or MALDI-TOF MS

• For phosphorylation analysis:

  • Phospho-enrichment using TiO₂ or IMAC before MS analysis

  • Phos-tag SDS-PAGE for mobility shift detection

  • In vitro kinase assays with recombinant kinases (consider HUNK based on ITM2A findings)

  • Metabolic labeling with ³²P for highly sensitive detection

• For proteolytic processing:

  • N-terminal sequencing to identify precise cleavage sites

  • MS/MS analysis of terminal peptides

  • Generation of cleavage site-specific antibodies

  • In vitro processing assays with purified proteases (e.g., furin)

• General PTM workflow:

  Step	Method	Consideration
  Enrichment	Immunoprecipitation with anti-Itm2c	Use detergent compatible with downstream analysis
  Fractionation	2D-PAGE or LC	Separate modified from unmodified forms
  Detection	MS/MS or Western blot	Use multiple complementary approaches
  Validation	Site-directed mutagenesis	Create non-modifiable mutants (e.g., T→A for phosphosites)
  Functional assessment	Cell-based assays	Compare WT vs. modification-deficient mutants

When designing experiments to study Itm2c PTMs, researchers should consider that recombinant protein produced in E. coli will lack eukaryotic PTMs, necessitating either mammalian expression systems or the use of enzymatic modification in vitro for functional studies.

What approaches are recommended for investigating the role of Itm2c in neurodegenerative pathways?

Investigating the role of Itm2c in neurodegenerative pathways requires a multi-dimensional approach that spans molecular, cellular, and in vivo levels of analysis. Based on what is known about Itm2c's interactions with APP and its potential role in protein processing, the following methodological approaches are recommended:

Molecular-level approaches:

• Protein-protein interaction studies:

  • Identify all Itm2c binding partners in neuronal cells using proximity labeling (BioID or APEX)

  • Perform Co-IP coupled with mass spectrometry to identify novel neurodegeneration-relevant interactors

  • Use surface plasmon resonance (SPR) to quantify binding affinities with known partners (e.g., APP)

• Structural studies:

  • Determine the 3D structure of Itm2c alone and in complex with APP fragments

  • Perform in silico docking studies to identify potential therapeutic binding sites

  • Conduct molecular dynamics simulations to understand conformational changes

Cellular-level approaches:

• Gain/loss-of-function studies:

  • Generate Itm2c knockout and overexpression neuronal cell lines using CRISPR/Cas9

  • Assess effects on APP processing, protein aggregation, and neuronal viability

  • Rescue experiments with mutant variants to identify critical residues/domains

• Cellular stress response:

  • Examine Itm2c's role in endoplasmic reticulum (ER) stress responses

  • Investigate effects on unfolded protein response (UPR) signaling

  • Assess impact on autophagy markers (LC3-II, p62) in neuronal models

• Trafficking studies:

  • Track Itm2c subcellular localization under normal and stress conditions

  • Examine co-localization with APP in different subcellular compartments

  • Study effects on lysosomal function and autophagy flux

In vivo approaches:

• Animal models:

  • Generate conditional Itm2c knockout or transgenic mice

  • Cross with established neurodegenerative disease models (APP/PS1, Tau models)

  • Examine effects on disease progression, pathology, and behavior

• Biomarker studies:

  • Develop assays to measure Itm2c and its processed fragments in CSF and plasma

  • Correlate levels with disease progression in patient samples

  • Evaluate potential as diagnostic or prognostic biomarkers

Therapeutic-oriented approaches:

• Small molecule screening:

  • Develop assays to identify modulators of Itm2c-APP interaction

  • Screen for compounds that enhance Itm2c's inhibitory effect on APP processing

  • Test promising candidates in cellular and animal models

• Experimental design considerations:

  Approach	Controls	Readouts	Limitations
  APP processing	APP only, catalytically inactive Itm2c	Aβ, sAPPα, CTFs	Overexpression artifacts
  Neuronal toxicity	Vector control, unrelated protein	Viability, neurite integrity	Cell type specificity
  Autophagy modulation	Rapamycin, Bafilomycin A1	LC3-II/LC3-I ratio, p62 levels	Indirect effects
  Animal studies	Wild-type littermates	Cognitive function, tissue pathology	Species differences

When investigating Itm2c in neurodegeneration, researchers should consider its potential role in multiple pathways beyond just APP processing, including autophagy, protein quality control, and membrane protein trafficking, all of which are implicated in neurodegenerative disease mechanisms.

How can researchers design experiments to elucidate the differential functions of Itm2c splice variants?

Designing experiments to investigate the differential functions of Itm2c splice variants requires careful planning to address the specific characteristics and potential functional differences between variants. Based on available information, human ITM2C has at least two splice variants: one with a deletion of amino acids 41-87 and another with a deletion of amino acids 151-187 . Similar variants might exist in rat Itm2c, though specific information was not provided in the search results.

Experimental design approaches:

• Expression construct preparation:

  • Generate expression vectors for each splice variant with identical tags

  • Create chimeric constructs to identify functional domains

  • Prepare domain-specific deletion mutants beyond known splice variants

  • Use inducible expression systems for temporal control

• Comparative localization studies:

  • Perform immunofluorescence microscopy with tagged variants

  • Use subcellular fractionation followed by Western blotting

  • Conduct live-cell imaging with fluorescently-tagged variants

  • Expected differences: Variants may localize to different cellular compartments based on missing domains

• Protein-protein interaction analysis:

  • Perform parallel Co-IP experiments with all variants

  • Use yeast two-hybrid or mammalian two-hybrid screens

  • Conduct BioID proximity labeling with each variant as bait

  • Compare interactome datasets using bioinformatics analysis

• Functional assays based on known Itm2c activities:

  Function	Assay	Expected Differences
  APP processing	Co-transfection with APP, measure Aβ by ELISA	Variants missing interacting domains may not inhibit processing
  SCG10 binding	In vitro binding assays, microtubule stability	Differential effects on neurite outgrowth
  Autophagy regulation	LC3-II/p62 Western blots, autophagy flux	Variants may show distinct effects on mTOR pathway activation
  Dimerization	Chemical crosslinking, native PAGE	Variants may show altered oligomerization properties

• Transcript-level analysis:

  • Develop splice variant-specific qRT-PCR assays

  • Examine tissue-specific and developmental expression patterns

  • Analyze regulation under stress conditions or disease states

  • RNA-seq analysis to identify co-regulated genes

• Structural analysis:

  • Conduct limited proteolysis to reveal structural differences

  • Perform CD spectroscopy to compare secondary structure elements

  • Use hydrogen-deuterium exchange mass spectrometry to identify exposed regions

  • Computational modeling to predict structural consequences of splice variations

• Functional rescue experiments:

  • Knock down endogenous Itm2c in cellular models

  • Rescue with individual splice variants

  • Compare ability to restore normal phenotypes

Analysis guidelines:

• Always express and analyze all variants in parallel under identical conditions

• Quantify expression levels to ensure comparable protein abundance

• Include appropriate controls (vector-only, unrelated protein)

• Consider the potential impact of epitope tags on function

• Validate key findings using multiple methodological approaches

This systematic approach will help researchers delineate the specific functional roles of different Itm2c splice variants, which may have important implications for understanding its physiological functions and potential involvement in pathological conditions.

What are the optimal experimental conditions for studying Itm2c's role in autophagy regulation?

Based on findings with the related protein ITM2A , Itm2c may also play a role in autophagy regulation. Designing optimal experimental conditions for studying this function requires careful consideration of multiple factors. Here are comprehensive guidelines for investigating Itm2c's potential role in autophagy:

Cell model selection:

• Recommended cell lines:

  • Neuronal cell lines (e.g., SH-SY5Y, Neuro-2a) - relevant to Itm2c's neuronal expression

  • SKBR-3 - used successfully for ITM2A autophagy studies

  • Primary neurons - for physiological relevance

  • HEK293T - for initial mechanistic studies due to ease of transfection

• Expression systems:

  • Transient transfection for acute effects

  • Stable cell lines with inducible expression for long-term studies

  • CRISPR/Cas9 knockout models with rescue experiments

Autophagy induction and monitoring:

• Autophagy induction protocols:

  • Nutrient starvation: EBSS medium (2-12 hours) - shown effective for ITM2A studies

  • mTOR inhibition: Rapamycin (100-500 nM, 4-24 hours)

  • ER stress: Tunicamycin (0.5-2 μg/mL, 6-24 hours)

• Autophagy flux monitoring:

  • LC3-II accumulation with/without lysosomal inhibitors (Bafilomycin A1, 100 nM)

  • p62/SQSTM1 degradation kinetics

  • Tandem mRFP-GFP-LC3 reporter for autolysosome formation

  • Transmission electron microscopy for autophagic vacuole visualization

Signaling pathway analysis:

• mTOR pathway monitoring:

  • Phospho-4EBP1 (T37/46) Western blotting - shown to be affected by ITM2A

  • Phospho-S6K (T389) analysis

  • Time course experiments (0-24 hours) to capture signaling dynamics

• AMPK pathway assessment:

  • Phospho-AMPK (T172) Western blotting

  • Phospho-ULK1 (S555) analysis

  • Comparative analysis with known AMPK activators (AICAR, metformin)

Experimental design recommendations:

Critical controls and validations:

• Expression verification:

  • Western blot to confirm Itm2c expression levels

  • Immunofluorescence to verify subcellular localization

• Autophagy-specific controls:

  • Rapamycin (positive control for autophagy induction)

  • Bafilomycin A1 (to block autophagy flux)

  • 3-Methyladenine (early autophagy inhibitor)

• Specificity controls:

  • Itm2c knockdown/knockout

  • Rescue with wild-type vs. mutant Itm2c

  • Comparison with related family members (ITM2A, ITM2B)

When designing these experiments, researchers should be aware that Itm2c's effects on autophagy might differ from those of ITM2A, and the experimental conditions may need to be optimized specifically for Itm2c. The mTOR-dependent mechanism identified for ITM2A provides a starting point but should not limit investigation of alternative pathways.

How does phosphorylation affect Itm2c function and what methods can detect these modifications?

Based on studies with the related protein ITM2A, which is phosphorylated at T35 by the kinase HUNK , phosphorylation may be an important regulatory mechanism for Itm2c function as well. Here's a comprehensive guide to studying potential phosphorylation of Itm2c:

Potential functional impacts of Itm2c phosphorylation:

• Protein-protein interactions:

  • Phosphorylation may modulate binding affinity to partners like APP or SCG10

  • May create or disrupt binding sites for phospho-binding domains (e.g., 14-3-3, SH2 domains)

• Subcellular localization:

  • Phosphorylation could affect trafficking between cellular compartments

  • May influence membrane insertion or retention

• Proteolytic processing:

  • Phosphorylation near cleavage sites might enhance or inhibit protease accessibility

  • Could affect furin-mediated processing (by analogy to human ITM2C processing)

• Signaling pathway integration:

  • May link Itm2c function to cellular stress responses

  • Could regulate autophagy in a stress-dependent manner (as seen with ITM2A)

Prediction of potential phosphorylation sites:

Sequence alignment with ITM2A suggests that Itm2c might have conserved phosphorylation sites. Bioinformatic tools like NetPhos, PhosphoSitePlus, and GPS can predict potential sites based on kinase recognition motifs.

Methods for detecting Itm2c phosphorylation:

• Mass spectrometry-based approaches:

  • Phosphopeptide enrichment using TiO₂ or IMAC

  • Parallel reaction monitoring (PRM) for targeted quantification

  • SILAC labeling for quantitative phosphoproteomics

  • Expected outcome: Identification of specific phosphorylated residues and their stoichiometry

• Biochemical detection methods:

  • Phos-tag SDS-PAGE for mobility shift detection

  • Phospho-specific antibodies (if available or custom-made)

  • ³²P metabolic labeling for high sensitivity detection

  • 2D gel electrophoresis to separate phospho-isoforms

• In vitro kinase assays:

  • Based on ITM2A findings, HUNK kinase should be tested

  • Recombinant Itm2c as substrate with purified kinases

  • ATP-γ-S labeling combined with PNBM alkylation (thiophosphate ester antibody detection)

  • Expected outcome: Identification of kinases that phosphorylate Itm2c in vitro

Functional validation of phosphorylation:

• Site-directed mutagenesis:

  • Generate phospho-deficient mutants (Ser/Thr → Ala)

  • Create phosphomimetic mutants (Ser/Thr → Asp/Glu)

  • Compare functional properties in:

    • APP processing assays

    • Autophagy modulation

    • Protein-protein interaction studies

• Kinase manipulation experiments:

  • Overexpress or inhibit candidate kinases (e.g., HUNK)

  • Monitor effects on Itm2c phosphorylation status

  • Assess functional consequences on Itm2c activities

• Physiological regulation:

  • Examine phosphorylation under stress conditions (starvation, ER stress)

  • Investigate cell cycle-dependent phosphorylation

  • Study tissue-specific phosphorylation patterns

Experimental design table for phosphorylation studies:

When studying Itm2c phosphorylation, researchers should consider that regulation might be context-specific and may vary between cell types, particularly between neuronal and non-neuronal cells. The finding that ITM2A phosphorylation increases during starvation suggests that nutrient status might be an important condition to examine for Itm2c as well.

What are the recommended controls when performing knockdown or knockout studies of Itm2c?

When designing knockdown or knockout studies to investigate Itm2c function, proper controls are essential to ensure experimental validity and interpretable results. Here's a comprehensive guide to controls for Itm2c loss-of-function studies:

Essential controls for Itm2c knockdown studies:

• Sequence-specific controls:

  • Non-targeting siRNA/shRNA with similar GC content

  • Multiple independent siRNA/shRNA sequences targeting different regions of Itm2c

  • Scrambled versions of the Itm2c-targeting sequences

  • Expected outcome: Consistent phenotypes across different targeting sequences indicate specificity

• Expression validation controls:

  • qRT-PCR to verify mRNA reduction (primers spanning different exons)

  • Western blot to confirm protein reduction

  • Immunofluorescence to assess cellular expression patterns

  • Expected validation threshold: >70% reduction in expression

• Rescue controls:

  • Re-expression of siRNA/shRNA-resistant Itm2c (with silent mutations)

  • Expression of orthologous Itm2c (e.g., mouse Itm2c in rat cells)

  • Domain-specific rescue with truncated Itm2c variants

  • Expected outcome: Reversal of knockdown phenotypes confirms specificity

Essential controls for Itm2c knockout studies:

• CRISPR/Cas9 controls:

  • Non-targeting gRNA with similar predicted off-target profile

  • Multiple independent gRNAs targeting different exons

  • Cas9-only expressing cells

  • Expected outcome: Consistent phenotypes across different gRNAs

• Clonal variation controls:

  • Analysis of multiple independent knockout clones

  • Pooled knockout populations to minimize clonal effects

  • Wild-type clones that went through the same selection process

  • Expected approach: At least 3 independent knockout clones should be characterized

• Genetic validation:

  • Genomic PCR and sequencing of the targeted locus

  • Western blot confirmation of protein absence

  • Off-target analysis through whole-genome sequencing or targeted sequencing of predicted sites

  • Expected validation: Confirmation of intended genetic modification without significant off-target effects

Functional controls:

• Pathway-specific controls:

  • For APP processing studies: APP knockout cells as negative control

  • For autophagy studies: Cells treated with rapamycin or starved in EBSS

  • For neurite outgrowth: SCG10 knockdown cells

• Related protein controls:

  • Expression analysis of ITM2A and ITM2B (potential compensation)

  • Double/triple knockdown with other ITM2 family members

  • Rescue experiments with other family members

Experimental design considerations:

Additional control considerations:

• Temporal controls:

  • Inducible knockdown/knockout systems to distinguish acute vs. chronic effects

  • Time course analysis after knockdown induction

  • Comparison with pharmacological inhibition (if available)

• Dosage controls:

  • Titration of siRNA/shRNA to achieve partial knockdown

  • Heterozygous knockout comparison

  • Correlation of phenotype strength with knockdown efficiency

• Cell type-specific controls:

  • Parallel knockdown in multiple relevant cell types

  • Comparison of effects in cells with high vs. low endogenous Itm2c expression

When interpreting results from Itm2c knockdown/knockout studies, researchers should be aware of potential compensation by other ITM2 family members (ITM2A, ITM2B) and consider the possibility that acute knockdown may produce different effects than stable knockout due to compensatory mechanisms.

How can researchers effectively study the dimerization properties of Itm2c?

Based on information that human ITM2C may exist as a dimer , studying the dimerization properties of rat Itm2c is an important aspect of understanding its functional mechanisms. Here's a comprehensive methodological approach to investigate Itm2c dimerization:

Biochemical methods for detecting Itm2c dimers:

• Chemical crosslinking:

  • Use membrane-permeable crosslinkers (DSS, BS3, formaldehyde)

  • Optimize crosslinker concentration and reaction time

  • Analyze by SDS-PAGE and Western blotting

  • Expected outcome: Detection of ~70-76 kDa bands (dimer) in addition to the 35-38 kDa monomer

• Native PAGE analysis:

  • Use mild detergents for membrane protein extraction (DDM, CHAPS)

  • Run samples without reducing agents and without boiling

  • Compare with and without crosslinking

  • Expected outcome: Higher molecular weight species in native conditions

• Size exclusion chromatography:

  • Use detergent-solubilized Itm2c

  • Compare elution profiles with known molecular weight standards

  • Analyze fractions by Western blotting

  • Expected outcome: Elution at positions consistent with monomer and dimer forms

• Analytical ultracentrifugation:

  • Perform sedimentation velocity experiments

  • Calculate molecular weight from sedimentation coefficients

  • Expected outcome: Multiple species corresponding to different oligomeric states

Biophysical approaches for characterizing dimerization:

• Förster resonance energy transfer (FRET):

  • Generate Itm2c constructs with compatible fluorophores (e.g., CFP/YFP pairs)

  • Perform both sensitized emission and acceptor photobleaching FRET

  • Include positive controls (known dimeric proteins) and negative controls

  • Expected outcome: FRET signal indicating close proximity (<10 nm) between tags

• Bioluminescence resonance energy transfer (BRET):

  • Tag Itm2c with Renilla luciferase and YFP

  • Measure energy transfer upon substrate addition

  • Create a saturation curve by varying acceptor:donor ratios

  • Expected outcome: Hyperbolic curve indicating specific interactions

• Single-molecule approaches:

  • Total internal reflection fluorescence (TIRF) microscopy with fluorescently-tagged Itm2c

  • Single-molecule tracking to detect co-diffusion of differentially labeled monomers

  • Expected outcome: Co-localization and co-diffusion of differently labeled Itm2c molecules

Structural determinants of dimerization:

• Domain mapping:

  • Generate truncation mutants lacking specific domains

  • Test dimerization capacity of each construct

  • Focus on transmembrane domain and BRICHOS domain

  • Expected outcome: Identification of domains necessary for dimer formation

• Site-directed mutagenesis:

  • Identify conserved residues likely to mediate protein-protein interactions

  • Create point mutations at these positions

  • Assess impact on dimerization using methods above

  • Expected outcome: Identification of specific residues critical for dimerization

• Computational prediction:

  • Use protein-protein docking software to predict dimerization interfaces

  • Molecular dynamics simulations to assess stability of predicted dimers

  • Guide experimental design based on in silico predictions

Functional significance of dimerization:

• Correlation with activity:

  • Compare functional activities of monomeric vs. dimeric forms

  • Assess APP processing inhibition by different oligomeric states

  • Examine autophagy regulation by monomers vs. dimers

  • Expected outcome: Determination if dimerization enhances, inhibits, or is required for function

• Inducible dimerization systems:

  • Create chimeric Itm2c fused to inducible dimerization domains (FKBP/FRB, Cry2)

  • Trigger dimerization with rapamycin or light

  • Monitor acute functional consequences

  • Expected outcome: Temporal correlation between forced dimerization and functional changes

Experimental design table:

When studying Itm2c dimerization, researchers should consider that membrane protein oligomerization may be influenced by the lipid environment, protein concentration, and cellular context. Therefore, complementary approaches in different systems (in vitro with purified protein, in cellular membranes, in live cells) will provide the most comprehensive understanding of Itm2c dimerization properties.

What are the methodological approaches for investigating Itm2c's potential role in disease models?

Investigating Itm2c's potential role in disease models requires a comprehensive strategy that spans from molecular mechanisms to in vivo disease models. Based on known functions of Itm2c and related proteins (including ITM2A's role in breast cancer ), here are methodological approaches for disease-focused Itm2c research:

Expression analysis in disease contexts:

• Tissue expression profiling:

  • Compare Itm2c expression levels in healthy vs. diseased tissues

  • Use qRT-PCR, Western blot, and immunohistochemistry

  • Analyze expression patterns in public databases (GEO, TCGA)

  • Expected approach: Paired analysis of same-patient normal/disease samples

• Single-cell transcriptomics:

  • Examine cell type-specific expression changes in disease models

  • Correlate with disease progression markers

  • Identify co-regulated gene networks

  • Expected outcome: Cell populations with altered Itm2c expression in disease states

• Biomarker potential assessment:

  • Develop assays for Itm2c detection in biological fluids

  • Compare levels between healthy subjects and disease cohorts

  • Correlate with disease severity and progression

  • Expected approach: ELISA or mass spectrometry-based quantification

Cellular disease models:

• Neurodegenerative disease models:

  • Express disease-associated APP mutations with/without Itm2c

  • Measure Aβ production, aggregation, and toxicity

  • Assess effects on tau pathology in relevant models

  • Expected outcome: Determination if Itm2c modifies APP processing or protects against toxicity

• Cancer models:

  • Based on ITM2A findings in breast cancer , investigate Itm2c in:

    • Proliferation assays (MTT, EdU incorporation)

    • Colony formation assays

    • Migration and invasion assays

    • Expected outcome: Assessment of tumor suppressor or oncogenic properties

• Autophagy-related disease models:

  • Study Itm2c in models of disorders with autophagy dysregulation

  • Assess ability to rescue defective autophagy phenotypes

  • Examine effects on protein aggregation clearance

  • Expected outcome: Determination if Itm2c modulation could restore normal autophagy

In vivo disease models:

• Neurodegenerative disease models:

  • Cross Itm2c transgenic or knockout mice with AD model mice

  • Assess effects on pathology (amyloid plaques, tangles)

  • Evaluate cognitive function using behavioral tests

  • Expected approach: Age-dependent analysis of pathology and behavior

• Cancer models:

  • Generate conditional Itm2c knockout in cancer-prone models

  • Create xenograft models with Itm2c-modulated cell lines

  • Analyze tumor growth, metastasis, and response to therapy

  • Expected outcome: In vivo validation of cellular findings

• Metabolic disease models:

  • Based on autophagy connection, examine Itm2c in:

    • Diet-induced obesity models

    • Diabetes models

    • Models of liver disease

  • Expected approach: Metabolic phenotyping with tissue-specific Itm2c modulation

Therapeutic targeting approaches:

• Target validation:

  • Structure-based design of Itm2c modulators

  • Screen for small molecules that promote Itm2c's protective functions

  • Develop antibodies that modulate Itm2c activity or processing

  • Expected outcome: Proof-of-concept compounds that modify Itm2c function

• Therapeutic delivery strategies:

  • Gene therapy approaches for Itm2c overexpression

  • siRNA delivery for Itm2c knockdown if pro-disease

  • Domain-specific peptide inhibitors or activators

  • Expected approach: Testing in cellular and animal models before clinical translation

Experimental design considerations:

When investigating Itm2c in disease models, researchers should consider:

• Disease-specific contexts where protein processing, autophagy, or membrane protein trafficking are implicated

• Potential protective vs. detrimental effects that may be context-dependent

• Interactions with established disease pathways and potential as a disease modifier

• Translational potential as a biomarker or therapeutic target

The promising findings with ITM2A as a prognostic marker and potential therapeutic target in breast cancer suggest that Itm2c may have similar roles in other diseases, particularly those involving autophagy dysregulation or protein processing defects.