Recombinant Human Integral membrane protein 2A (ITM2A)

What is the structural organization of human ITM2A protein?

ITM2A is a 263-amino acid type II transmembrane protein that belongs to the BRICHOS superfamily. The protein contains four defined structural regions: a hydrophobic domain, a linker region, an extracellular BRICHOS domain (approximately 100 amino acids) thought to have chaperone function, and an intracellular C-terminal domain. The transmembrane topology of ITM2A places the N-terminus in the cytoplasm and the C-terminus in the extracellular or luminal space, which is critical for its biological functions .

What are the commonly used expression systems for recombinant human ITM2A production?

For recombinant expression of human ITM2A, researchers typically employ mammalian expression systems such as HEK293T cells, which allow for proper post-translational modifications. When studying phosphorylation states of ITM2A, such as the T35 residue, mammalian systems are particularly valuable. For protein interaction studies, fusion proteins like GST-ITM2A may be expressed in bacterial systems and purified for in vitro assays. When designing expression constructs, researchers should consider whether to express full-length ITM2A or specific domains, depending on the research question .

How can phosphorylation status of ITM2A at T35 be effectively detected in experimental settings?

To detect phosphorylation of ITM2A at T35, researchers should employ a multi-faceted approach. Initial identification may use mass spectrometry of immunoprecipitated ITM2A, which can reveal phosphorylation sites as demonstrated in published studies. For site-specific analysis, researchers can generate phospho-specific antibodies against the T35 site or use phospho-mimetic (T35D/E) and non-phosphorylatable (T35A) mutants. For functional validation of phosphorylation, in vitro kinase assays using purified components (such as HUNK kinase and recombinant ITM2A) should be performed. Western blotting with phospho-specific antibodies and phos-tag SDS-PAGE can offer quantitative assessment of phosphorylation states under different experimental conditions .

What experimental approaches best evaluate the role of ITM2A in autophagy regulation?

To evaluate ITM2A's role in autophagy, researchers should employ complementary approaches. First, autophagy flux can be monitored using fluorescent reporters like mRFP-GFP-LC3, which allows distinction between autophagosomes (yellow puncta) and autolysosomes (red-only puncta) through confocal microscopy. Western blotting analysis of autophagy markers (LC3-II conversion and p62 degradation) provides quantitative measurement of autophagy activity. Transmission electron microscopy (TEM) enables visualization of autophagic vacuoles at ultrastructural level. Additionally, researchers should assess mTOR pathway activity by measuring phosphorylation of downstream targets like 4EBP1 at T37/46. To establish causality, ITM2A knockdown or overexpression experiments, combined with autophagy inducers (starvation) or inhibitors (bafilomycin A1), will determine whether ITM2A-mediated effects are autophagy-dependent .

What are the critical considerations when designing site-directed mutagenesis experiments for ITM2A phosphorylation sites?

When designing site-directed mutagenesis experiments for ITM2A phosphorylation sites, researchers should consider several critical factors. First, conservation analysis across species should be performed to confirm evolutionary significance of the phosphorylation site, as demonstrated for the T35 residue which is highly conserved across multiple species. Non-phosphorylatable mutants (typically T→A substitutions) and phosphomimetic mutants (T→D or T→E) should both be generated for comprehensive functional analysis. Expression vectors should contain appropriate tags (Flag, HA, or GFP) for detection and immunoprecipitation while ensuring tags do not interfere with ITM2A localization or function. Functional readouts should include not only phosphorylation status but also downstream effects such as autophagy induction and cell proliferation, as T35A mutation has been shown to abolish ITM2A's autophagy-inducing and growth-inhibitory effects .

How does ITM2A expression influence breast cancer cell proliferation in experimental models?

ITM2A exhibits significant anti-proliferative effects in breast cancer experimental models through multiple mechanisms. Overexpression of ITM2A in breast cancer cell lines significantly inhibits cell proliferation as measured by standard assays including MTT assay, colony formation assay, and EdU incorporation assay. The growth inhibitory effect of ITM2A is partially dependent on its phosphorylation status at T35, as the T35A mutant exhibits significantly reduced anti-proliferative capacity compared to wild-type ITM2A. Mechanistically, ITM2A-mediated growth inhibition appears to be linked to its role in promoting autophagy, which may contribute to tumor suppression in certain contexts. These findings suggest that ITM2A functions as a tumor suppressor in breast cancer, and its downregulation may contribute to enhanced cancer cell proliferation and tumor progression .

How can ITM2A expression be effectively restored in cancer cells for therapeutic applications?

For therapeutic restoration of ITM2A expression in cancer cells, researchers should consider multiple approaches. Gene delivery systems using viral vectors (adenoviral, lentiviral) can efficiently reintroduce ITM2A expression in cancer cells with low endogenous levels. For enhanced stability and function, phosphomimetic ITM2A variants (T35D/E) may provide superior therapeutic efficacy compared to wild-type protein, as phosphorylation at T35 is critical for ITM2A's autophagy-inducing and growth-inhibitory functions. Combination therapies targeting both ITM2A restoration and the HUNK kinase pathway could synergistically enhance therapeutic outcomes. For clinical translation, researchers should develop biomarker strategies to identify patients most likely to benefit from ITM2A-targeted therapies, focusing on those with low ITM2A expression, particularly in PR-positive or HER2-enriched breast cancer subtypes .

How does ITM2A interact with the mTOR signaling pathway to regulate autophagy?

ITM2A regulates autophagy through interactions with the mTOR signaling pathway, a master regulator of cellular metabolism. Experimental evidence demonstrates that ITM2A overexpression significantly inhibits mTOR activity, as indicated by reduced phosphorylation of downstream targets such as 4EBP1 at T37/46 residues. This inhibition of mTOR signaling promotes autophagy induction, characterized by increased LC3-II levels, decreased p62 levels, and enhanced formation of autophagosomes and autolysosomes. The inhibitory effect of ITM2A on mTOR is dependent on its phosphorylation status at T35, as the T35A mutation abolishes this inhibitory effect, resulting in restored mTOR activity (elevated p-4EBP1) and reduced autophagy. While the exact molecular mechanism by which ITM2A inhibits mTOR remains to be fully elucidated, these findings establish ITM2A as a novel regulator of autophagy through an mTOR-dependent mechanism .

What is the functional significance of the HUNK-ITM2A interaction in cellular processes?

The interaction between HUNK (Hormonally Up-regulated Neu-associated Kinase) and ITM2A represents a significant regulatory mechanism with functional implications for cellular processes, particularly in cancer contexts. HUNK directly phosphorylates ITM2A at T35, as demonstrated through in vitro kinase assays using purified recombinant proteins. This phosphorylation is functionally significant, as it is required for ITM2A-mediated autophagy induction and growth inhibition. The HUNK-ITM2A axis potentially connects HER2 signaling with autophagy regulation, as HUNK is highly expressed in HER2-positive breast cancers and promotes survival in response to lapatinib treatment. Both proteins independently correlate with breast cancer patient survival, suggesting their interaction constitutes an important regulatory node. While ITM2A functions as a tumor suppressor, HUNK has been implicated in promoting HER2-induced mammary tumorigenesis, indicating a complex relationship that warrants further investigation .

How can protein-protein interaction networks involving ITM2A be comprehensively mapped?

To comprehensively map ITM2A protein-protein interaction networks, researchers should implement a multi-omics approach. Affinity purification combined with mass spectrometry (AP-MS) using tagged-ITM2A as bait can identify direct binding partners under various cellular conditions. Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling can capture transient or weak interactions within the cellular vicinity of ITM2A. For domain-specific interactions, researchers should employ yeast two-hybrid screening with individual ITM2A domains (transmembrane, BRICHOS, C-terminal). Validation of identified interactions requires co-immunoprecipitation, GST pull-down assays, and fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) for live-cell visualization. Functional characterization should follow, focusing on how specific interactions affect ITM2A phosphorylation, subcellular localization, stability, and downstream functions like autophagy regulation or growth inhibition .

What are the optimal conditions for performing in vitro kinase assays with ITM2A and HUNK?

For optimal in vitro kinase assays with ITM2A and HUNK, researchers should follow a precise protocol. The kinase source should be immunoprecipitated Flag-HUNK from transfected cells, ideally comparing different cellular conditions (nutrient-rich versus EBSS starvation) that may affect kinase activity. Substrate preparation requires purified recombinant GST-ITM2A (wild-type and T35A mutant) expressed in bacterial systems and purified to high homogeneity. Reaction conditions should include 25 mM HEPES (pH 7.5), 10 mM MgCl₂, 50 mM NaCl, 1 mM DTT, 0.5 mg/ml BSA, 250 μM Na₃VO₄, 50 mM NaF, 1 mM EDTA, 0.2 mM AMP, and 0.2 mM non-radioactive ATP, incubated at 30°C for 30 minutes. Phosphorylation detection can utilize phospho-specific antibodies or radiolabeled ATP (γ-³²P-ATP) followed by autoradiography. Control reactions should include kinase-dead HUNK variants and non-phosphorylatable ITM2A mutants (T35A) .

How can autophagy flux be accurately measured in the context of ITM2A studies?

For accurate measurement of autophagy flux in ITM2A studies, researchers should employ multiple complementary approaches. The gold standard approach uses tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) reporters, which enable distinction between autophagosomes (yellow puncta) and autolysosomes (red-only puncta) due to GFP quenching in acidic environments. Quantification should include both the number of puncta per cell and the ratio of red-only to yellow puncta. This should be complemented with Western blot analysis of LC3-I to LC3-II conversion and p62 degradation, with samples treated with and without lysosomal inhibitors (bafilomycin A1, chloroquine) to distinguish between autophagy induction and blockade. Transmission electron microscopy provides ultrastructural confirmation of autophagic vacuoles. When studying ITM2A variants, all methods should be applied comparatively to wild-type ITM2A, phospho-mutants (T35A), and vector controls under both basal and starvation-induced conditions .

What statistical approaches are most appropriate for analyzing ITM2A expression data in clinical samples?

For analyzing ITM2A expression data in clinical samples, researchers should apply rigorous statistical methodologies tailored to the specific dataset characteristics. For diagnostic and prognostic value assessment, Receiver Operating Characteristic (ROC) curve analysis with calculation of Area Under the Curve (AUC) provides quantitative evaluation of ITM2A as a biomarker. Kaplan-Meier survival analysis with log-rank tests should be used to correlate ITM2A expression levels with patient outcomes, stratifying patients into high and low expression groups based on median expression or optimal cutoff values determined by ROC analysis. For correlation with clinical parameters (age, tumor stage, receptor status), Mann-Whitney tests are appropriate for continuous variables and Chi-square or Fisher's exact tests for categorical variables. Multivariate Cox regression analysis should be performed to determine whether ITM2A is an independent prognostic factor. All statistical analyses should be conducted using established software such as GraphPad Prism or R, with p-values <0.05 considered statistically significant .

How might ITM2A function be exploited for development of novel cancer therapeutics?

Exploiting ITM2A function for novel cancer therapeutics presents several promising strategies. Given ITM2A's tumor suppressor role and frequent downregulation in breast cancer, gene therapy approaches could restore ITM2A expression in tumors with low endogenous levels. Alternatively, small molecule screens could identify compounds that either upregulate endogenous ITM2A expression or mimic its downstream effects on autophagy and mTOR inhibition. Since ITM2A phosphorylation at T35 by HUNK is critical for its function, developing peptide mimetics of the phosphorylated region could potentially recapitulate ITM2A activity. For precision medicine applications, ITM2A expression levels could serve as a biomarker to stratify patients for specific treatments, particularly for hormone therapy in PR-positive breast cancers. Combination therapies targeting both the HUNK-ITM2A axis and established pathways (HER2, mTOR) might yield synergistic anti-tumor effects, especially in aggressive breast cancer subtypes .

What are the unexplored aspects of ITM2A biology that warrant further investigation?

Several unexplored aspects of ITM2A biology warrant further investigation to expand our understanding of this protein's functions. While ITM2A's role in autophagy and breast cancer has been established, its functions in other cancer types and normal tissues remain largely uncharacterized. The BRICHOS domain of ITM2A is thought to have chaperone functions, but specific client proteins and folding activities require detailed biochemical analysis. The membrane topology and trafficking of ITM2A between cellular compartments (endoplasmic reticulum, Golgi, plasma membrane) remain poorly understood. Beyond phosphorylation at T35, other potential post-translational modifications (glycosylation, ubiquitination) and their functional significance need exploration. The evolutionary conservation of ITM2A across species suggests important biological functions that extend beyond currently known roles. Finally, the relationship between ITM2A and its family members (ITM2B, ITM2C) deserves comparative analysis to determine unique and overlapping functions in health and disease .

How does ITM2A function compare across different tissue and cancer types?

Comparative analysis of ITM2A function across tissues and cancer types represents an important research direction. While evidence from breast cancer studies shows ITM2A downregulation correlates with poor prognosis, systematic investigation across cancer types using TCGA and other database analyses would reveal whether this pattern is universal or cancer-specific. Tissue-specific expression patterns of ITM2A should be mapped in normal human tissues to establish baseline expression levels and potentially identify tissues where ITM2A plays crucial physiological roles. Different cancer subtypes may exhibit varying degrees of ITM2A dysregulation; preliminary evidence suggests particularly significant correlations in HER2-enriched breast cancers, warranting similar subtype analysis in other cancers. The HUNK-ITM2A phosphorylation axis should be examined across cancer types to determine if this regulatory mechanism is conserved or context-dependent. This comparative approach would ultimately inform the therapeutic potential of targeting ITM2A across different cancer contexts .