Recombinant Mouse Receptor expression-enhancing protein 4 (Reep4)

What is the molecular structure of mouse REEP4 and how does it compare to human REEP4?

Mouse REEP4 belongs to the REEP protein family characterized by hydrophobic domains that facilitate membrane interactions, particularly with the endoplasmic reticulum (ER). The protein contains specific domains that contribute to its ability to maintain high ER membrane curvature, which is essential during cellular processes such as mitosis. Research has confirmed that REEP4 is required for high ER membrane curvature during cell division .

When comparing mouse and human REEP4, researchers should conduct sequence alignment analysis to identify conserved functional domains. While the specific sequence homology isn't detailed in the available data, membrane-associated proteins like REEP4 typically show conservation in functional domains across mammalian species. For experimental design, it's crucial to consider potential structural differences that might affect antibody recognition or functional assays when translating between mouse models and human applications.

What are the primary cellular functions of REEP4 in mouse models?

REEP4 serves several critical functions in mouse cellular processes. Most prominently, it plays an essential role in endoplasmic reticulum organization during cell division. REEP4 is strongly implicated in vital cellular processes such as cell division and protein synthesis, with significant involvement in maintaining proper ER membrane architecture during mitosis .

To investigate these functions experimentally, researchers can utilize techniques such as fluorescently-tagged recombinant REEP4 for live-cell imaging during mitosis, or conduct loss-of-function studies followed by rescue experiments with recombinant protein. When designing functional studies, consider that REEP4's effects may vary across different cell types depending on their ER structure and division rates. Quantitative assays measuring ER morphology, membrane curvature, and cell division progression can provide valuable metrics for assessing recombinant REEP4 functionality.

What is the subcellular localization pattern of REEP4 and how can it be visualized in mouse cells?

REEP4 demonstrates a distinct subcellular localization pattern, with predominant expression in the cytosol and nucleoplasm . This dual localization pattern suggests it may perform different functions depending on its cellular compartmentalization. As a membrane-associated protein, REEP4 interacts extensively with the endoplasmic reticulum, particularly during cell division processes.

For visualization studies, researchers can employ immunofluorescence using validated antibodies against mouse REEP4 or utilize tagged recombinant constructs. For optimal results, combine confocal microscopy with super-resolution techniques such as STORM or STED to accurately resolve REEP4's association with ER membranes. Co-staining with markers for different ER domains, nuclear envelope, and cell division machinery can provide contextual information about REEP4's dynamic localization during cellular processes. When using recombinant tagged proteins, validate that the tag doesn't interfere with localization by comparing to endogenous protein patterns.

How does REEP4 expression vary across different mouse tissues and developmental stages?

A comprehensive understanding of REEP4 expression patterns across tissues and developmental stages is essential for contextualizing experimental findings. To establish these patterns, researchers should employ multiple complementary approaches including quantitative PCR, western blotting, and immunohistochemistry across a panel of mouse tissues and developmental timepoints.

For developmental studies, examine REEP4 expression from embryonic through postnatal stages, paying particular attention to tissues with high cell division rates where REEP4's role in mitosis may be most significant. In adult tissues, correlate expression levels with tissue-specific ER characteristics and proliferation rates. This baseline expression data provides crucial context for interpreting experiments with recombinant REEP4 and helps identify physiologically relevant models for specific research questions.

What factors regulate REEP4 expression in mouse models, and how can this knowledge inform experimental design?

Understanding the regulatory mechanisms controlling REEP4 expression provides valuable insights for experimental design with recombinant proteins. To identify regulatory factors, researchers should analyze the REEP4 promoter region for transcription factor binding sites, conduct reporter assays to measure promoter activity under various conditions, and examine expression changes in response to cellular stressors or signaling pathway activation.

Given REEP4's role in cell division, investigate potential cell cycle-dependent regulation through synchronized cell populations and cell cycle inhibitors. When designing overexpression studies with recombinant REEP4, consider how the experimental system might trigger compensatory changes in endogenous regulatory mechanisms. Including time-course analyses and measuring both recombinant and endogenous protein levels can help identify such regulatory feedback effects that might influence experimental outcomes.

How does REEP4 expression correlate with disease states in mouse models, particularly in cancer?

Research has demonstrated significant correlations between REEP4 expression and disease states, particularly in cancer models. Human studies have shown that REEP4 expression is significantly upregulated in kidney clear cell carcinoma (KIRC) tissues compared to normal tissues, with higher expression levels positively correlating with tumor malignancy . These findings suggest potential applications for mouse models in investigating REEP4's role in cancer progression.

For experimental approaches, researchers should establish baseline REEP4 expression in relevant mouse cancer models compared to healthy tissues. Correlation analyses between REEP4 levels and tumor characteristics such as proliferation rate, invasiveness, and treatment response can identify potential mechanistic relationships. The observed relationship between REEP4 and cancer immunotherapy response in human studies suggests that mouse models investigating immune system interactions would be particularly valuable .

What expression systems yield optimal results for recombinant mouse REEP4 production, and what methodological considerations are important?

The choice of expression system significantly impacts the yield, folding, and functionality of recombinant mouse REEP4. For membrane-associated proteins like REEP4, mammalian expression systems often provide the most authentic post-translational modifications and folding environment. HEK293 or CHO cells are particularly suitable for maintaining proper membrane protein conformation.

Methodologically, consider using inducible expression systems to control expression levels, as high REEP4 concentration may disrupt ER membranes in host cells. For the expression construct, include a cleavable affinity tag (His, FLAG, or GST) positioned to minimize interference with membrane interactions. Codon optimization for the host system and inclusion of chaperone co-expression may improve yields. Carefully optimize induction conditions, temperature, and duration to balance between yield and proper folding, with lower temperatures (28-30°C) often favoring correct folding over maximum expression.

What purification strategies maintain structural integrity and functionality of recombinant mouse REEP4?

Purifying functional recombinant REEP4 requires strategies that preserve its native structure and membrane-interacting properties. Begin with affinity chromatography using the incorporated tag, optimizing buffer conditions with appropriate detergents that solubilize REEP4 while maintaining structural integrity. For membrane proteins like REEP4, mild non-ionic detergents such as DDM, CHAPS, or Brij-35 often provide good results.

Following initial capture, consider size exclusion chromatography to separate properly folded monomers from aggregates, which is particularly important for membrane-associated proteins. Throughout purification, monitor protein quality using techniques such as circular dichroism to assess secondary structure and thermal shift assays to evaluate stability. Functional validation through membrane binding assays or interaction studies with known partners provides the ultimate confirmation of proper structure. For long-term storage, evaluate stabilizing additives and determine optimal protein concentration to prevent aggregation.

What quality control measures should be implemented to validate recombinant mouse REEP4 for functional studies?

Comprehensive quality control is essential for ensuring recombinant REEP4 accurately represents the native protein in functional studies. Begin with basic biochemical characterization including SDS-PAGE for purity assessment (aim for >90%), western blotting with specific antibodies, and mass spectrometry to confirm protein identity and detect any modifications or truncations.

For structural validation, employ multiple complementary approaches: circular dichroism to assess secondary structure elements, limited proteolysis to evaluate domain folding, and thermal shift assays to determine stability. Functional validation should include membrane binding assays measuring REEP4's ability to associate with and modify ER-like membranes, particularly assessing membrane curvature effects. Interaction studies with known binding partners provide another functional validation approach. Finally, cellular assays demonstrating the recombinant protein's ability to rescue REEP4 knockout phenotypes offer the most physiologically relevant validation.

How can recombinant mouse REEP4 be utilized to study endoplasmic reticulum dynamics during cell division?

Recombinant mouse REEP4 offers powerful tools for investigating ER dynamics during cell division. For live imaging approaches, fluorescently tagged recombinant REEP4 enables visualization of ER reorganization during mitosis in real-time. Optimally, use photoactivatable or photoconvertible tags for pulse-chase experiments that track specific ER domains through the division process.

For mechanistic studies, compare wild-type recombinant REEP4 with domain mutants to identify regions essential for ER membrane remodeling during mitosis. Combine these tools with super-resolution microscopy techniques such as STED or STORM to resolve detailed structural changes. In reconstitution assays, purified recombinant REEP4 added to artificial membrane systems can directly demonstrate membrane deformation capabilities. These approaches provide complementary perspectives on REEP4's role in maintaining high ER membrane curvature during mitosis, a function previously confirmed in research .

What approaches can be used to identify and validate novel REEP4 interaction partners in mouse cells?

Identifying REEP4's interaction network provides crucial insights into its cellular functions. For unbiased discovery of novel interaction partners, employ proximity labeling approaches such as BioID or APEX, where enzymatically active tags on recombinant REEP4 biotinylate nearby proteins that can then be purified and identified by mass spectrometry. These methods are particularly valuable for membrane-associated proteins like REEP4 where traditional co-immunoprecipitation may disrupt weak or detergent-sensitive interactions.

For targeted validation of specific interactions, use multiple complementary approaches: co-immunoprecipitation with recombinant tagged REEP4, pulldown assays with purified recombinant protein, and in situ techniques such as proximity ligation assay or FRET to confirm interactions in intact cells. Functional validation of identified interactions can be achieved through mutational analysis of interaction interfaces and phenotypic analysis of interaction-deficient mutants. When designing these experiments, consider REEP4's dual localization in cytosol and nucleoplasm , as interaction partners may differ between compartments.

How can recombinant mouse REEP4 contribute to understanding cancer progression and potential therapeutic approaches?

Research has demonstrated significant associations between REEP4 expression and cancer progression, particularly in kidney clear cell carcinoma (KIRC) . Recombinant mouse REEP4 provides valuable tools for investigating these associations mechanistically. In cell culture models, comparing the effects of wild-type versus mutant recombinant REEP4 on proliferation, migration, and invasion can identify functional domains critical for cancer-promoting activities.

For immunotherapy applications, the observed relationship between REEP4 expression and immunotherapy response can be investigated using recombinant REEP4 in co-culture systems with immune cells. REEP4's potential as a therapeutic target can be assessed by developing and screening inhibitors against the recombinant protein, then validating promising candidates in cellular and animal models. Additionally, the observed correlation between REEP4 expression and drug sensitivity suggests applications in developing predictive biomarkers for treatment response, with recombinant REEP4 serving as a standard for assay development.

What statistical approaches are most appropriate for analyzing experiments using recombinant mouse REEP4?

Selecting appropriate statistical methods is essential for robust interpretation of REEP4 experiments. For dose-response studies with recombinant REEP4, employ regression analysis to establish EC50/IC50 values with confidence intervals rather than testing isolated concentrations. When comparing effects across multiple experimental conditions, use ANOVA with appropriate post-hoc tests rather than multiple t-tests to control family-wise error rates.

For time-course experiments, repeated measures ANOVA or mixed-effects models offer more statistical power than endpoint analyses. When integrating multiple data types (e.g., expression levels, functional outcomes, and clinical correlates), multivariate approaches such as principal component analysis or partial least squares regression can identify patterns not apparent in univariate analyses. Power analysis before experimentation ensures sufficient sample sizes for detecting biologically meaningful effects, particularly important when using precious recombinant protein resources.

How should researchers address contradictory findings between recombinant protein studies and genetic approaches to REEP4 function?

Contradictions between recombinant protein and genetic approaches often provide valuable insights rather than simply representing experimental failures. When facing such contradictions, systematically evaluate several potential explanations: differences in protein levels (overexpression versus physiological), timing effects (acute versus chronic manipulation), compensatory mechanisms activated in genetic models but not acute recombinant protein studies, or technical issues with recombinant protein functionality.

Resolution strategies include titrating recombinant protein concentrations to physiological levels, using inducible genetic systems that mirror the timing of recombinant protein experiments, employing genome editing to introduce precise mutations rather than complete knockouts, and careful validation of recombinant protein functionality. Integrating multiple complementary approaches provides the most robust understanding—combining observations from recombinant protein studies, genetic manipulations, and endogenous protein analyses to triangulate REEP4's true biological functions.

What controls are essential for properly interpreting experiments with recombinant mouse REEP4?

Robust experimental design with appropriate controls is critical for valid interpretation of recombinant REEP4 studies. Include multiple control types: negative controls (inactive mutant versions of REEP4, heat-denatured protein, or irrelevant proteins of similar size) to distinguish specific from non-specific effects; vehicle controls that undergo identical processing to the recombinant protein preparation to identify buffer or contaminant effects; and dose controls with titration series to distinguish physiological from pharmacological effects.

For cellular experiments, include both untransfected cells and cells expressing control proteins to account for non-specific effects of protein overexpression. When possible, validate key findings using complementary approaches such as genetic knockdown/knockout followed by rescue with recombinant protein. For immunological detection, include controls for antibody specificity such as REEP4-depleted samples. These comprehensive controls enable confident attribution of observed effects specifically to REEP4 function rather than experimental artifacts.

How can post-translational modifications of mouse REEP4 be studied, and what is their functional significance?

Post-translational modifications (PTMs) likely play critical roles in regulating REEP4 function, particularly during cell division. For comprehensive PTM identification, analyze purified recombinant and endogenous REEP4 using mass spectrometry approaches optimized for each modification type (phosphorylation, ubiquitination, acetylation, etc.). Compare modification patterns between different cellular contexts, particularly across cell cycle stages, to identify regulatory PTMs.

To determine functional significance, generate recombinant REEP4 variants with mutations at modification sites: non-modifiable mutations (e.g., S→A for phosphorylation sites) and modification-mimicking mutations (e.g., S→D/E for phosphorylation). Compare these variants in functional assays measuring membrane binding, protein interactions, and ER remodeling activities. In cellular contexts, assess the ability of these variants to rescue REEP4 knockout phenotypes, particularly focusing on cell division processes where REEP4's role in maintaining ER membrane curvature is essential .

What transgenic mouse models would be most informative for studying REEP4 function in vivo?

Developing transgenic mouse models provides powerful systems for investigating REEP4 function in physiological contexts. Consider several complementary approaches: conditional knockout models using Cre-lox systems enable tissue-specific and temporal control of REEP4 deletion, avoiding potential embryonic lethality if REEP4 is essential for development; knock-in models expressing tagged REEP4 at endogenous levels allow visualization and biochemical studies without overexpression artifacts; and point mutation knock-in models targeting specific functional domains provide precise mechanistic insights.

For cancer research applications, combine REEP4 modifications with established tumor models (e.g., crossing with KIRC mouse models) to investigate its role in cancer progression. For studying REEP4's role in immunotherapy response , develop models with modified immune compartments. When designing these models, incorporate features facilitating experimental analysis, such as reporter genes linked to REEP4 expression or inducible systems for temporal control of transgene activation.

How might REEP4 function in specialized cell types with unique endoplasmic reticulum structures?

REEP4's role in ER membrane organization may take on specialized functions in cell types with unique ER structures. To investigate these specialized functions, select cell types with distinctive ER architectures such as pancreatic acinar cells (extensive rough ER), plasma cells (expanded ER for antibody production), or neurons (specialized ER in dendrites and axons). Compare REEP4 localization, interaction partners, and loss-of-function phenotypes across these specialized cells.

Methodologically, combine high-resolution imaging of fluorescently tagged recombinant REEP4 with electron microscopy to correlate protein localization with ultrastructural features. Use cell-type-specific conditional knockout models followed by rescue with wild-type or mutant recombinant REEP4 to determine domain requirements in specialized contexts. These approaches can reveal how REEP4's general function in maintaining ER membrane curvature during mitosis may be adapted to serve specialized functions in differentiated cells with unique ER architectures.

How conserved is REEP4 structure and function across species, and what insights does this provide for mouse model relevance?

Evolutionary conservation analysis provides crucial context for extrapolating findings from mouse REEP4 studies to other species, particularly humans. Conduct comprehensive sequence comparison across mammalian species, focusing separately on different protein domains to identify differentially conserved regions. Beyond sequence comparison, structural prediction algorithms can identify conservation of three-dimensional features despite sequence divergence.

To experimentally assess functional conservation, perform cross-species complementation studies where recombinant REEP4 from different species is tested for its ability to rescue phenotypes in REEP4-depleted cells. These approaches help establish which aspects of mouse REEP4 research are likely translatable to human biology. Regions showing high conservation across species (particularly in the membrane-interacting domains) likely represent functionally critical regions that should be prioritized in mechanistic studies and therapeutic targeting efforts.

What can be learned from comparing REEP4 with other REEP family members in mouse models?

Comparative analysis across the REEP protein family provides valuable insights into both shared and unique functions of REEP4. Begin with comprehensive expression profiling of all REEP family members across tissues and developmental stages to identify patterns of co-expression or complementary expression. In tissues where multiple REEPs are expressed, investigate potential functional redundancy through single and combinatorial knockdown/knockout approaches.

For mechanistic understanding, compare recombinant REEP proteins in biochemical and cellular assays to identify shared versus specialized functions. Domain-swapping experiments between REEP4 and other family members can pinpoint regions responsible for functional differences. This comparative approach helps contextualize REEP4-specific findings within the broader REEP family functions, particularly distinguishing between REEP4's unique role in maintaining ER membrane curvature during mitosis and functions it may share with other family members.

How do environmental factors and experimental conditions affect REEP4 expression and function in mouse models?

Understanding environmental and experimental influences on REEP4 is essential for robust experimental design and data interpretation. Systematically investigate how factors such as cellular stress, nutrient availability, temperature, and pH affect REEP4 expression and localization. Given REEP4's role in ER membrane organization, particular attention should be paid to conditions that affect ER homeostasis, such as unfolded protein response activation.

For experimental design considerations, determine how cell culture conditions (2D versus 3D culture, substrate stiffness, cell density) influence REEP4 function, particularly in processes related to cell division. In animal models, evaluate whether housing conditions, diet, or microbiome composition affect REEP4 expression or function. These analyses help establish standardized conditions for REEP4 research and identify potential environmental factors that might influence its biological roles in both physiological and pathological contexts.

What emerging technologies could advance our understanding of mouse REEP4 structure and function?

Emerging technologies offer exciting opportunities to address longstanding questions about REEP4. Cryo-electron microscopy could resolve REEP4's structure in membrane contexts, particularly identifying how it induces membrane curvature. For dynamic studies, lattice light-sheet microscopy with adaptive optics would enable long-term visualization of REEP4 during cell division with minimal phototoxicity, providing unprecedented insights into its temporal dynamics.

Genome editing advances using prime editing or base editing could generate precise modifications of endogenous REEP4, avoiding overexpression artifacts. Single-cell multi-omics approaches combining transcriptomics, proteomics, and metabolomics could reveal how REEP4 function varies across cell populations and states. Organoid technologies offer platforms for studying REEP4 in physiologically relevant 3D tissue contexts, particularly valuable for cancer-related studies given REEP4's association with tumor progression .

What are the most promising therapeutic applications stemming from mouse REEP4 research?

Research on mouse REEP4 suggests several promising therapeutic directions. The association between REEP4 expression and cancer progression, particularly in kidney clear cell carcinoma , indicates potential as a biomarker for prognosis or treatment selection. Developing inhibitors targeting REEP4 or its critical interactions could provide novel therapeutic approaches for cancers where REEP4 is overexpressed.

For immunotherapy applications, the observed relationship between REEP4 expression and immunotherapy response suggests potential as a predictive biomarker for treatment selection. Further research could identify whether modulating REEP4 might enhance immunotherapy effectiveness. Additionally, the correlation between REEP4 expression and sensitivity to specific anticancer drugs indicates applications in personalized medicine approaches, where REEP4 expression might guide treatment selection among available options.

How might interdisciplinary approaches advance mouse REEP4 research beyond current limitations?

Interdisciplinary collaboration offers powerful approaches to overcome current limitations in REEP4 research. Combining structural biology with computational modeling could predict how REEP4 interacts with membranes and identify potential binding sites for small molecule modulators. Integrating cell biology with biophysics approaches could quantitatively measure how REEP4 affects membrane mechanical properties during cell division.

Systems biology approaches analyzing REEP4 within broader protein networks could contextualize its functions within cellular signaling pathways. Collaborations between basic scientists and clinical researchers could accelerate translation of findings from mouse models to human applications, particularly in cancer contexts where REEP4 shows prognostic associations . Machine learning approaches applied to integrated datasets could identify previously unrecognized patterns in REEP4 function and regulation across cellular contexts and disease states.