RAET1E Human, IgG-His

What is RAET1E and what molecular family does it belong to?

RAET1E is a member of the MHC class I family and belongs to the RAET1 gene family. It encodes surface proteins that function as ligands for the NKG2D receptor expressed on immune cells. The RAET1 genes are positioned in a cluster on chromosome 6q24.2-q25.3 and encode glycoproteins containing extracellular alpha-1 and alpha-2 domains . RAET1E is also known by several synonyms including Lymphocyte Effector Toxicity Activation Ligand, RAE-1-Like Transcript 4, NKG2DL4, ULBP4, and NKG2D Ligand 4 .

Unlike standard MHC class I molecules, RAET1E lacks the membrane-proximal Ig-like alpha-3 domain. The recombinant human RAET1E protein consists of 204 amino acids (residues 31-225) with a molecular mass of approximately 23.4 kDa, though it appears at 28-40 kDa on SDS-PAGE due to glycosylation .

How does RAET1E differ structurally from other RAET1 family members?

RAET1E has a distinctive structure compared to other RAET1 family members. While most RAET1 proteins are anchored to the cell membrane via glycosylphosphatidylinositol (GPI) linkage, RAET1E and RAET1G have type I membrane-spanning sequences at their C-termini . This structural difference may confer unique functional properties and cellular localization patterns to these proteins.

The amino acid sequence of RAET1E contains specific domains that facilitate its interaction with the NKG2D receptor, enabling it to deliver activating signals to natural killer (NK) cells and cytotoxic T lymphocytes . This interaction is critical for immune surveillance against transformed or stressed cells.

How is RAET1E expression regulated at the transcriptional level?

RAET1E expression is regulated by multiple transcriptional mechanisms:

• E2F transcription factors directly activate RAET1 gene expression, linking RAET1E expression to cell cycle entry and proliferation . This explains why RAET1E is often upregulated in rapidly dividing cells, including cancer cells.

• The RAET1E promoter contains specific regulatory elements that respond to different cellular conditions. Studies have identified a major transcription start site (TSS-1) in the RAET1E gene, and variations in the promoter region can affect transcription efficiency .

• Promoter functionality studies using dual-luciferase reporter assays have demonstrated that sequence variations in the RAET1E promoter region can significantly impact its expression. Specifically, four FVB sequence variations present in the first 426 bp of the promoter and the C57 variant of SNP rs50817078 (JMRv10073) have been shown to alter RAET1E expression levels .

What cellular conditions induce RAET1E expression?

RAET1E expression is induced under several cellular conditions:

• Cellular Proliferation: RAET1E is strongly induced in proliferating cells. In primary fibroblast cultures, RAET1E expression is detected within 2 days of culture initiation and reaches a plateau after 6 days .

• Growth Factor Stimulation: Growth factors present in serum or defined growth factors like EGF can induce RAET1E expression. This induction is directly linked to the proliferative effect of these factors rather than other serum components .

• Signaling Pathway Activation: Several signaling pathways regulate RAET1E expression. Inhibitors of cyclin-dependent kinases (Roscovitine), the PI3K-mTOR pathway (LY294002, Rapamycin), and the MAPK pathway (SB202190) strongly block RAET1E induction, suggesting these pathways are essential for RAET1E expression .

• Tissue Repair Processes: RAET1E expression is induced in cells involved in healing skin wounds, suggesting a role for RAET1E in tissue repair mechanisms .

What is the relationship between E2F transcription factors and RAET1E expression?

E2F transcription factors play a central role in regulating RAET1E expression:

• E2F directly activates transcription of RAET1 family genes, including RAET1E .

• Since E2F transcription factors regulate cell cycle entry, this mechanism links RAET1E expression to cellular proliferation, explaining why RAET1E is upregulated in rapidly dividing cells like cancer cells .

• This regulation appears to be specific to certain NKG2D ligands. While RAET1 family members (including RAET1E) are strongly induced in proliferating primary fibroblasts, other NKG2D ligands like MULT1 and H60b are induced only modestly, if at all .

• The E2F-mediated transcriptional activation of RAET1E is likely coordinated with other stress responses through posttranscriptional regulation mechanisms .

How does RAET1E interact with the NKG2D receptor to mediate immune responses?

RAET1E functions as a ligand for the NKG2D receptor, which is expressed on various immune cells including natural killer (NK) cells, CD8+ T cells, and γδ T cells . This interaction triggers several important immune responses:

• NK Cell Activation: When RAET1E binds to NKG2D on NK cells, it delivers activating signals that can overcome inhibitory signals, leading to NK cell cytotoxicity against target cells expressing RAET1E .

• Tumor Immune Surveillance: RAET1E advances tumor immune surveillance by inducing the growth of anti-tumor cytotoxic lymphocytes . This makes RAET1E an important component of the body's natural defense against cancer.

• Balanced Immune Response: The interaction between RAET1E and NKG2D is part of a complex system where the balance between inhibitory and activating signals determines the outcome of target recognition by immune cells .

• Affinity and Specificity: RAET1E binds specifically to NKG2D with high affinity, allowing for effective immune cell recognition of stressed or transformed cells that upregulate RAET1E expression .

What is the significance of RAET1E being a transmembrane protein rather than GPI-anchored?

Most RAET1 family proteins are anchored to the cell membrane via GPI linkage, but RAET1E and RAET1G are unique in having type I membrane-spanning sequences at their C-termini . This structural difference has several important implications:

• Cellular Localization: Transmembrane anchoring may result in different membrane localization compared to GPI-anchored proteins, potentially affecting interactions with other membrane components and receptors.

• Signal Transduction: The transmembrane domain may confer unique signal transduction capabilities that are not possible with GPI-anchored proteins.

• Protein Stability and Turnover: The different anchoring mechanism may affect protein stability, internalization, and turnover rates at the cell surface.

• Immune Evasion Resistance: Transmembrane anchoring might provide resistance to certain viral immune evasion strategies that target GPI-anchored NKG2D ligands.

• Functional Diversity: The existence of both GPI-anchored and transmembrane RAET1 proteins suggests a diversification of functional roles, potentially allowing for a more nuanced immune response to different types of cellular stress .

How is RAET1E involved in atherosclerosis pathogenesis?

Research has identified RAET1E as a novel atherosclerosis modifier gene . Evidence supporting this role includes:

• Altered Expression: Studies have shown altered RAET1E gene expression underlying the Ath11 10b locus, a genetic region associated with atherosclerosis susceptibility .

• Endothelial Expression: RAET1E is expressed in lesional aortic endothelial cells and macrophage-rich regions, suggesting a direct role in the atherosclerotic process .

• Genetic Confirmation: The creation of RAET1E transgenic mice has confirmed the role of RAET1E in atherosclerosis, making it one of the few successful identifications of an atherosclerosis gene using the quantitative trait locus (QTL) strategy in complex diseases .

• Molecular Basis: The molecular basis for altered aortic RAET1E expression and its atherosclerosis phenotype has been revealed to be a mutation in the transcription initiation region of the RAET1E gene .

What is the role of RAET1E in tumor immunology?

RAET1E plays a significant role in tumor immunology through several mechanisms:

• Immune Surveillance: RAET1E acts as a ligand for the NKG2D receptor, delivering signals to NK cells and promoting tumor immune surveillance by inducing the growth of anti-tumor cytotoxic lymphocytes .

• Stress-Induced Expression: As a stress-induced protein, RAET1E can be upregulated in cancer cells, marking them for recognition and elimination by NKG2D-bearing immune cells .

• Proliferation Link: The regulation of RAET1E by E2F transcription factors links its expression to cellular proliferation, which is often dysregulated in cancer cells. This mechanism potentially enables the immune system to identify rapidly dividing cancer cells .

• Immune Evasion Counteraction: The induction of RAET1E in proliferating cells may represent an evolutionary adaptation to counteract immune evasion strategies employed by cancer cells .

What are the optimal handling and storage conditions for recombinant RAET1E Human, IgG-His?

For optimal handling and storage of recombinant RAET1E protein:

• Storage Buffer: RAET1E protein solution (0.5mg/ml) contains Phosphate Buffered Saline (pH 7.4) and 10% glycerol .

• Long-term Storage: For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) to enhance stability .

• Temperature: While not explicitly stated in the search results, recombinant proteins are typically stored at -80°C for long-term storage or -20°C for short-term storage.

• Freeze-Thaw Cycles: Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity .

• Working Solutions: When preparing working solutions, it's advisable to dilute the stock solution in appropriate buffers immediately before use.

What experimental approaches can be used to assess RAET1E promoter functionality?

The dual-luciferase reporter assay has been effectively used to assess RAET1E promoter functionality:

• Cloning Strategy: RAET1E promoter fragments from different genomic DNA sources (e.g., FVB and C57) upstream of the major aortic transcription start site (TSS-1) are cloned into luciferase reporter vectors such as pGl4.11[luc2CP] .

• Site-Directed Mutagenesis: Site-directed mutagenesis can be used to introduce specific sequence variations into the promoter fragments to assess their impact on transcriptional activity .

• Cell Lines: Multiple cell lines can be used for transfection experiments, including C57SV002, BalbcSV006, and NIH3T3 cells, to account for cell type-specific effects .

• Co-Transfection: The expression vectors are co-transfected with a control vector (e.g., pGl4.74[hRluc/TK]) to normalize for transfection efficiency .

• Measurement: Luciferase activities are measured using dual-luciferase reporter assay systems, allowing for quantitative assessment of promoter functionality under different conditions or with different genetic variants .

What cell models are appropriate for studying RAET1E expression and function?

Several cell models have been effectively used to study RAET1E expression and function:

• Primary Fibroblasts: Primary cultures of fibroblasts prepared from various tissues (tail, peritoneal wall, ear) have shown robust induction of RAET1E expression within days of culture initiation, making them excellent models for studying RAET1E regulation .

• Cell Lines: Various cell lines have been used for studying RAET1E promoter functionality, including:

  • C57SV002 (Jackson Laboratory)

  • BalbcSV006 (Jackson Laboratory)

  • NIH3T3 cells (ATCC)

• Insect Cells: Sf9 insect cells have been used for the production of recombinant RAET1E protein, suggesting they can properly express and process the protein .

• Primary Cultures from Different Mouse Strains: Primary fibroblasts from different mouse strains (B6, BALB/c, 129/J) can be used to study strain-specific differences in RAET1E expression .

• In Vivo Models: For studying RAET1E in physiological contexts, embryonic brain cells and cells in healing skin wounds have shown RAET1E expression, making these valuable models for in vivo studies .

How can RAET1E be used as a target for immunotherapeutic approaches?

RAET1E offers several promising avenues for immunotherapeutic approaches:

• NK Cell Activation: Since RAET1E binding to NKG2D activates NK cells and promotes tumor immune surveillance, strategies to enhance this interaction could potentially boost anti-tumor immune responses .

• Cancer Immunotherapy: Understanding the regulation of RAET1E by E2F transcription factors provides insights for developing therapies that could selectively enhance RAET1E expression in cancer cells, making them more visible to the immune system .

• Atherosclerosis Treatment: Given RAET1E's role as an atherosclerosis modifier gene, targeting RAET1E or its regulatory pathways might offer novel approaches for atherosclerosis treatment .

• Diagnostic Applications: The selective expression of RAET1E in certain disease contexts could be exploited for diagnostic purposes, potentially through imaging techniques using labeled antibodies against RAET1E.

• Combination Therapies: Understanding how RAET1E interacts with the immune system could inform the development of combination therapies that enhance immune recognition of diseased cells while targeting other aspects of disease pathology.

What are the key experimental challenges in studying RAET1E-NKG2D interactions?

Researchers studying RAET1E-NKG2D interactions face several experimental challenges:

• Protein Stability: Maintaining the native conformation of RAET1E during purification and experimental procedures can be challenging, potentially affecting interaction studies.

• Glycosylation Effects: The glycosylation state of RAET1E significantly affects its molecular weight and potentially its function, requiring careful consideration in experimental design .

• Binding Affinity Measurement: Accurately measuring the binding affinity between RAET1E and NKG2D requires specialized techniques such as surface plasmon resonance or bio-layer interferometry.

• Functional Readouts: Developing appropriate functional readouts for RAET1E-NKG2D interactions in different cellular contexts can be complex, as the outcome may depend on various factors including the presence of other activating or inhibitory receptors.

• Specificity Controls: Given the existence of multiple NKG2D ligands, ensuring specificity in RAET1E-focused studies requires careful controls and potentially the use of blocking antibodies or gene knockdown/knockout approaches.

How does RAET1E function compare across different species?

RAET1E belongs to a family of proteins that show interesting evolutionary patterns:

• Human vs. Mouse: In mice, the homologous proteins are members of the retinoic acid early inducible gene 1 (RAE-1; α–ε) subfamily, which share functional similarities with human RAET1E in binding to NKG2D and activating immune cells . Both human RAET1E and mouse RAE-1 proteins are induced in proliferating cells and regulated by E2F transcription factors .

• Structural Differences: While the basic function of binding to NKG2D is conserved, there may be species-specific differences in structure, expression patterns, and regulatory mechanisms that reflect evolutionary adaptations to different immune challenges.

• Expression Patterns: Studies in mice have shown that RAE-1ε expression is induced in primary cultures, embryonic brain cells in vivo, and cells in healing skin wounds . Comparative studies of human RAET1E expression in similar contexts would be valuable for understanding cross-species similarities and differences.

What are the most promising future research directions for RAET1E?

Several promising research directions for RAET1E include:

• Detailed Structural Studies: More detailed structural characterization of RAET1E, particularly its interaction with NKG2D, could provide insights for the design of therapeutic agents that modulate this interaction.

• Role in Other Diseases: While RAET1E has been implicated in atherosclerosis and cancer, its potential role in other inflammatory or immune-related diseases remains to be fully explored.

• Regulatory Network Mapping: Further elucidation of the complex regulatory networks controlling RAET1E expression, including transcriptional, post-transcriptional, and post-translational mechanisms, would enhance our understanding of how RAET1E expression is modulated in different physiological and pathological contexts.

• Therapeutic Development: Development of targeted approaches to modulate RAET1E expression or function for therapeutic purposes in cancer, atherosclerosis, or other diseases represents an exciting frontier in RAET1E research.

• Single-Cell Analysis: Application of single-cell technologies to study RAET1E expression and function at the individual cell level could reveal new insights into its role in heterogeneous cell populations and tissues.