Recombinant Mouse Type-1 angiotensin II receptor-associated protein (Agtrap)

Advanced Research Questions

• What experimental approaches best demonstrate Agtrap's interaction with AT1 receptor in vitro?

  Several complementary methods can be employed to demonstrate and characterize the Agtrap-AT1 receptor interaction:

  1. Co-immunoprecipitation (Co-IP): Using specific antibodies against either Agtrap or AT1 receptor to pull down protein complexes, followed by western blot analysis to detect the interacting partner.

  2. GST fusion protein pull-down assays: Recombinant GST-tagged Agtrap can be used to pull down AT1 receptor from cell lysates, confirming direct physical interaction .

  3. Surface Plasmon Resonance (SPR): Provides quantitative binding kinetics (kon, koff) and affinity (KD) measurements. This method has successfully demonstrated Agtrap-RACK1 interaction and can be adapted for AT1 receptor studies .

  4. Yeast two-hybrid screening: Useful for initial identification of interaction partners and mapping interaction domains .

  5. FRET/BRET assays: For studying interactions in living cells, where AT1 receptor and Agtrap are tagged with compatible fluorophores.

  6. Proximity Ligation Assay (PLA): Allows visualization of protein interactions in situ with high specificity and sensitivity.

  When designing these experiments, it's critical to include appropriate controls such as non-interacting protein pairs and to confirm findings using multiple independent techniques.

• How does Agtrap expression affect AT1 receptor signaling pathways in cardiovascular disease models?

  Agtrap modulates AT1 receptor signaling through several mechanisms that impact cardiovascular pathophysiology:

  1. Receptor Internalization: Agtrap enhances AT1 receptor internalization, thereby attenuating angiotensin II signaling duration and intensity.

  2. MAPK Pathway Modulation: Agtrap negatively regulates angiotensin II-induced MAPK activation, which is crucial in cardiovascular remodeling and hypertrophy .

  3. NF-κB Signaling: Agtrap influences NF-κB pathway activation, affecting inflammatory responses in cardiovascular tissues .

  4. Calcium Signaling: Agtrap modifies angiotensin II-induced calcium mobilization, impacting vascular smooth muscle contraction.

  In mouse models of hypertension and heart failure, Agtrap overexpression has demonstrated cardioprotective effects, reducing cardiac hypertrophy and improving vascular function. Conversely, Agtrap knockdown exacerbates angiotensin II-induced cardiovascular damage.

  For studying these pathways, researchers should consider using:

  • Phospho-specific antibodies for MAPK pathway components

  • Luciferase reporter assays for NF-κB activation

  • Calcium imaging in cardiomyocytes or vascular smooth muscle cells

  • Echocardiography and blood pressure measurements in transgenic mouse models

• What is the role of Agtrap in immune cell function and how does this impact inflammatory disease models?

  Emerging evidence indicates Agtrap plays significant roles in immune regulation:

  1. Immune Cell Infiltration: Agtrap expression positively correlates with infiltration of CD8+ T cells, CD4+ T cells, B cells, macrophages, dendritic cells, and neutrophils in certain disease models .

  2. T-Cell Exhaustion: Agtrap levels show significant correlation with T-cell exhaustion biomarkers, suggesting a potential immunomodulatory function .

  3. Inflammatory Signaling: Through its effects on NF-κB and MAPK pathways, Agtrap influences pro-inflammatory cytokine production.

  These findings suggest Agtrap may serve as an immunomodulatory target in inflammatory conditions. In mouse models of inflammatory diseases, researchers should consider:

  • Flow cytometric analysis of immune cell populations in Agtrap knockout versus wild-type mice

  • Cytokine profiling using multiplex assays or ELISA

  • Single-cell RNA sequencing to identify Agtrap-dependent transcriptional programs in specific immune cell subsets

  • Adoptive transfer experiments to distinguish cell-intrinsic versus extrinsic effects

• How can CRISPR-Cas9 be used to study Agtrap function in mouse models?

  CRISPR-Cas9 technology offers powerful approaches for investigating Agtrap function:

  1. Knockout Models: Complete deletion of Agtrap gene to study loss-of-function phenotypes

     • Design multiple sgRNAs targeting early exons

     • Verify knockout by genomic sequencing, RT-PCR, and western blotting

     • Assess cardiovascular phenotypes (blood pressure, cardiac function, vascular reactivity)

  2. Knock-in Models: Introduction of specific mutations or tags

     • Create fluorescent fusion proteins for live imaging

     • Introduce disease-associated mutations to study pathological mechanisms

     • Add epitope tags for improved detection and purification

  3. Conditional Knockouts: Tissue-specific deletion using Cre-loxP system

     • Combine with tissue-specific promoters (e.g., Tie2-Cre for endothelial cells, α-MHC-Cre for cardiomyocytes)

     • Enable temporal control using inducible systems (e.g., tamoxifen-inducible CreERT2)

  4. Transcriptional Modulation: CRISPRa/CRISPRi for activation or repression without altering the genomic sequence

  For effective CRISPR-Cas9 editing of Agtrap, recommended sgRNA design should target conserved functional domains, particularly the transmembrane regions or the AT1 receptor interaction domain. Off-target effects should be minimized through careful sgRNA design and verified by whole-genome sequencing.

Methodological Questions

• What are the optimal conditions for expressing soluble Recombinant Mouse Agtrap in E. coli?

  For optimal expression of soluble Recombinant Mouse Agtrap in E. coli:

  1. Expression Vector and Strain Selection:

     • Vectors: pET series (particularly pET28a) for T7 promoter-driven expression

     • Strains: BL21(DE3), Rosetta(DE3), or Arctic Express for difficult-to-express proteins

  2. Expression Conditions:

     • Induction: 0.1-0.5 mM IPTG at OD600 of 0.6-0.8

     • Temperature: Lower temperature (16-18°C) overnight induction improves solubility

     • Media: Enriched media (2YT or TB) supplemented with glucose (0.5-1%)

  3. Solubility Enhancement Strategies:

     • Fusion tags: N-terminal GST tag and His tag combination improves solubility and enables dual purification

     • Chaperone co-expression: GroEL/GroES system to assist proper folding

     • Addition of 5-10% glycerol to lysis buffer

  4. Purification Approach:

     • Two-step purification: Ni-NTA affinity followed by glutathione-sepharose chromatography

     • Buffer optimization: PBS pH 7.4 with 10% glycerol, 1 mM DTT, and protease inhibitors

     • Optional tag removal: TEV protease cleavage site between protein and tags

  Using these optimized conditions, yields of 10-15 mg/L of culture with >90% purity after two-step purification can be achieved.

• How can the functional activity of purified Recombinant Mouse Agtrap be verified?

  Verifying the functional activity of purified Recombinant Mouse Agtrap should involve multiple complementary approaches:

  1. Binding Assays:

     • Surface Plasmon Resonance (SPR) to measure binding kinetics to AT1 receptor

     • ELISA-based binding assays using immobilized AT1 receptor

     • Pull-down assays with AT1 receptor-expressing cell lysates

  2. Cellular Functional Assays:

     • AT1 receptor internalization assay using fluorescently-labeled angiotensin II

     • Inhibition of angiotensin II-induced calcium flux in AT1 receptor-expressing cells

     • Suppression of angiotensin II-stimulated ERK1/2 phosphorylation

  3. Activity Benchmarks:

     • For binding assays: KD value of 10-100 nM range indicates properly folded protein

     • For functional assays: IC50 values of 0.1-1 μM for inhibition of angiotensin II signaling

  4. Controls:

     • Positive control: Native Agtrap isolated from mouse tissues

     • Negative control: Heat-denatured recombinant Agtrap

     • Specificity control: Unrelated transmembrane protein of similar size

  Assessment of these parameters provides comprehensive validation of recombinant Agtrap functionality before application in complex experimental systems.

• What techniques can be used to study Agtrap's role in NF-κB and MAPK signaling pathways?

  To investigate Agtrap's role in NF-κB and MAPK signaling pathways:

  1. Phosphorylation Analysis:

     • Western blotting with phospho-specific antibodies (p-ERK1/2, p-p38, p-JNK, p-IκB)

     • Phospho-proteomics for global pathway analysis

     • Time-course experiments (0-60 minutes post-stimulation) to capture signaling dynamics

  2. Transcriptional Activity Assays:

     • Luciferase reporter assays for NF-κB activation

     • qRT-PCR for target gene expression (IL-6, TNF-α, IL-1β)

     • Chromatin immunoprecipitation (ChIP) to assess NF-κB binding to target promoters

  3. Protein-Protein Interaction Studies:

     • Co-immunoprecipitation of Agtrap with pathway components

     • Proximity ligation assay for in situ interaction visualization

     • FRET/BRET assays for real-time interaction monitoring

  4. Functional Manipulation:

     • Gain/loss-of-function approaches using recombinant protein, siRNA, or CRISPR

     • Pharmacological inhibitors of specific pathway components

     • Domain mapping to identify critical regions for pathway regulation

  5. In Vivo Validation:

     • Tissue-specific knockout models

     • Ex vivo tissue analysis for pathway activation

     • Disease model phenotyping (inflammation, fibrosis, hypertrophy)

  These approaches should be applied in relevant cell types, including vascular smooth muscle cells, cardiomyocytes, and immune cells where Agtrap function is physiologically important .

• How can Recombinant Mouse Agtrap be effectively labeled for tracking in cellular and in vivo experiments?

  Several labeling strategies can be employed for tracking Recombinant Mouse Agtrap:

  1. Genetic Fusion Tags:

     • Fluorescent proteins (GFP, mCherry, mScarlet) for live-cell imaging

     • Split fluorescent proteins for detecting protein-protein interactions

     • Enzyme tags (HaloTag, SNAP-tag) for pulse-chase labeling with membrane-permeable dyes

  2. Chemical Labeling:

     • Amine-reactive dyes (NHS esters) targeting lysine residues

     • Thiol-reactive probes (maleimides) for specific cysteine labeling

     • Click chemistry approaches using azide/alkyne tags with minimal structural perturbation

  3. Site-Specific Labeling:

     • Introduce unique cysteines at specific positions for targeted labeling

     • Enzymatic approaches like sortase-mediated ligation

     • Unnatural amino acid incorporation for bioorthogonal chemistry

  4. For In Vivo Tracking:

     • Radiolabeling with 125I or 18F for PET/SPECT imaging

     • Near-infrared fluorescent dyes for deeper tissue penetration

     • Conjugation to nanoparticles for multimodal imaging

  For cellular localization studies, it's crucial to verify that labeling doesn't affect Agtrap's membrane localization, interaction with AT1 receptor, or signaling properties. Control experiments should include comparison with unlabeled protein function and immunostaining of native protein to confirm physiological distribution patterns.

• What are the emerging therapeutic applications of targeting Agtrap in cardiovascular and inflammatory diseases?

  Research on Agtrap as a therapeutic target is revealing several promising applications:

  1. Cardiovascular Applications:

     • Hypertension management: Modulating Agtrap levels could provide alternative approaches to traditional angiotensin receptor blockers

     • Cardiac hypertrophy: Agtrap overexpression shows anti-hypertrophic effects in preclinical models

     • Vascular remodeling: Targeting Agtrap to inhibit pathological smooth muscle proliferation

  2. Inflammatory Disease Applications:

     • Hepatocellular carcinoma: AGTRAP is identified as a potential biomarker and therapeutic target

     • Immune modulation: Targeting Agtrap to influence T-cell exhaustion in chronic inflammatory conditions

     • Fibrotic disorders: Exploiting Agtrap's role in TGF-β signaling to attenuate fibrosis

  3. Therapeutic Modalities Under Investigation:

     • Small molecule modulators of Agtrap-AT1 receptor interaction

     • Peptide mimetics that enhance Agtrap's inhibitory effects

     • Gene therapy approaches for tissue-specific Agtrap overexpression

     • RNA therapeutics (siRNA, antisense oligonucleotides) for targeted knockdown

  4. Challenges in Therapeutic Development:

     • Cell type-specific functions requiring targeted delivery approaches

     • Potential compensatory mechanisms in chronic modulation

     • Off-target effects on other signaling pathways

  These emerging applications highlight the importance of continued basic research on Agtrap biology to fully understand its therapeutic potential and develop effective targeting strategies.