Recombinant Pongo abelii Bombesin receptor-activated protein C6orf89 homolog

How does this protein relate to the bombesin receptor family?

The bombesin receptor family consists of three G protein-coupled receptors (GPCRs): neuromedin B (NMB) receptor (BB1), gastrin-releasing peptide (GRP) receptor (BB2), and the orphan receptor bombesin receptor subtype 3 (BRS-3 or BB3) . The Pongo abelii Bombesin receptor-activated protein C6orf89 homolog was originally identified as a potential interacting partner of BRS-3, though subsequent verification studies using techniques like yeast two-hybrid and co-immunoprecipitation did not confirm a direct interaction .

Despite its name, current research suggests that this protein may function independently of bombesin receptors. The bombesin receptor family itself is involved in numerous physiological processes including itch perception, CNS functions, immune responses, and gastrointestinal activities .

What are the optimal conditions for expression and purification of recombinant Pongo abelii Bombesin receptor-activated protein?

The recombinant full-length protein is typically expressed in E. coli with an N-terminal His tag . For optimal expression and purification:

• Expression system: E. coli is the preferred host for recombinant production .

• Purification method: Ni-NTA affinity chromatography leveraging the His tag .

• Storage conditions:

  • Store at -20°C/-80°C upon receipt

  • For extended storage, maintain at -20°C or -80°C

  • Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

  • The shelf life of lyophilized form is approximately 12 months at -20°C/-80°C, while the liquid form has a shelf life of about 6 months .

• Reconstitution protocol:

  • Briefly centrifuge the vial prior to opening

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) and aliquot for long-term storage

What are the challenges in ensuring proper folding of the recombinant protein and how can they be addressed?

Ensuring proper folding of the recombinant Pongo abelii Bombesin receptor-activated protein presents several challenges:

• Membrane protein characteristics: As a type II membrane protein with a transmembrane domain, the hydrophobic regions can cause aggregation during expression in E. coli .

• Methodological approaches to improve folding:

  • Optimized expression temperature: Lowering the expression temperature to 16-18°C can reduce inclusion body formation

  • Solubility enhancers: Adding solubility tags like SUMO or MBP in addition to the His tag

  • Co-expression with chaperones: Utilizing E. coli strains engineered to overexpress molecular chaperones

  • Detergent screening: For membrane-associated regions, screening various detergents (DDM, LDAO, etc.) during purification

• Verification of proper folding:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Size exclusion chromatography to evaluate monodispersity

  • Limited proteolysis to probe for compact folded domains

  • Functional assays to confirm biological activity

How can researchers investigate the cellular localization and trafficking of Bombesin receptor-activated protein?

To investigate cellular localization and trafficking:

• Immunofluorescence microscopy:

  • Express the protein with fluorescent tags (GFP, mCherry) or use antibodies against the His tag or the protein itself

  • Co-stain with organelle markers (ER, Golgi, endosomes, lysosomes)

  • Use live-cell imaging to track protein movement in real-time

• Subcellular fractionation:

  • Separate cellular components using differential centrifugation

  • Analyze fractions by Western blotting to detect the protein of interest

  • Compare distribution patterns with known organelle markers

• Protein trafficking kinetics:

  • Employ pulse-chase experiments with metabolic labeling

  • Use photoactivatable or photo-switchable fluorescent tags

  • Apply pharmacological inhibitors of specific trafficking pathways to determine routes

• Interaction with trafficking machinery:

  • Perform co-immunoprecipitation to identify binding partners

  • Use proximity-dependent labeling methods (BioID, APEX) to identify proteins in close proximity

  • Apply yeast two-hybrid screening to discover novel interacting proteins

Previous research indicates that this protein primarily localizes to cytoplasmic compartments rather than the cell membrane, despite having a predicted transmembrane domain .

What methodologies are effective for studying interactions between this protein and ATG5 in autophagy regulation?

The interaction between Bombesin receptor-activated protein homolog and ATG5 in autophagy regulation can be studied using these approaches:

• Interaction validation methods:

  • Co-immunoprecipitation (Co-IP) with tagged versions of both proteins

  • Proximity ligation assay (PLA) to visualize protein interactions in situ

  • FRET or BRET analysis to demonstrate physical proximity in living cells

  • Pull-down assays using purified recombinant proteins to test direct interaction

• Functional autophagy assays:

  • LC3 puncta formation assays in cells with manipulated protein levels

  • Autophagic flux measurements using tandem fluorescent-tagged LC3 (mRFP-GFP-LC3)

  • p62/SQSTM1 degradation assays by Western blotting

  • Transmission electron microscopy to visualize autophagosomes

• Domain mapping:

  • Create truncation mutants to identify interaction domains

  • Site-directed mutagenesis of key residues to pinpoint critical interaction sites

  • Peptide array screening to identify specific binding motifs

Research has shown that BRAP homolog negatively regulates autophagy through interaction with ATG5, and lack of this protein leads to enhanced autophagy activity in mouse lung tissues and isolated fibroblasts .

How can researchers effectively use animal models to study the role of Bombesin receptor-activated protein homolog in fibrotic diseases?

Based on previous research with BC004004 knockout mice (lacking the mouse homolog of BRAP), the following approaches are recommended:

• Bleomycin-induced lung fibrosis model:

  • Administer bleomycin (1-3 mg/kg) intratracheally to induce pulmonary fibrosis

  • Compare wild-type and knockout mice for differences in:

    • Histopathological changes (H&E, Masson's trichrome staining)

    • Hydroxyproline content (quantitative measure of collagen)

    • Expression of fibrosis markers (α-SMA, collagen I/III, fibronectin)

    • Inflammatory cytokine profiles (TGF-β1, IL-1β, IL-6, TNF-α)

  • Isolate and culture primary fibroblasts to assess proliferation rates and collagen production

• Assessment of autophagy activity:

  • Analyze LC3-I to LC3-II conversion by Western blotting

  • Immunostaining for autophagy markers in tissue sections

  • Electron microscopy to quantify autophagosomes

  • Monitor autophagic flux using chloroquine or bafilomycin A1

• Rescue experiments:

  • Administer recombinant protein to knockout mice

  • Use adenoviral vectors for targeted re-expression

  • Apply autophagy inhibitors to determine if enhanced autophagy mediates the resistance to fibrosis

Research has shown that BC004004-/- mice exhibited attenuated pulmonary injury and less pulmonary fibrosis following bleomycin treatment, suggesting that lacking BRAP homologous protein leads to protection against fibrosis .

What methodological approaches should be employed to investigate the role of Bombesin receptor-activated protein homolog in inflammatory skin conditions?

Based on studies using the imiquimod (IMQ)-induced psoriasis-like model in BC004004 knockout mice:

• Experimental design for psoriasis-like inflammation:

  • Apply IMQ cream (5%) daily to the shaved back skin for 7 days

  • Monitor and score skin inflammation parameters:

    • Erythema (redness)

    • Scaling

    • Thickness (measured by caliper)

    • Transepidermal water loss (TEWL)

  • Document the temporal pattern of inflammation onset and resolution

• Molecular and cellular analysis:

  • Histological assessment (H&E, immunohistochemistry)

  • Flow cytometry to quantify immune cell infiltration

  • RT-qPCR to measure expression of inflammatory cytokines

  • ELISA to determine serum levels of:

    • IL-17A (marker of Th17 activation)

    • TSLP (thymic stromal lymphopoietin)

    • IL-1β, TGF-β1, IL-23

• In vitro mechanistic studies:

  • siRNA knockdown of BRAP in keratinocyte cell lines

  • Measurement of cytokine release (particularly TSLP)

  • Co-culture systems with immune cells to study epithelial-immune cell interactions

  • Chromatin immunoprecipitation (ChIP) to investigate transcriptional regulation of cytokine genes

Research has demonstrated that BC004004-/- mice develop skin lesions with earlier and more acute onset but quicker remission, accompanied by altered cytokine expression patterns .

A table summarizing different experimental systems for studying Bombesin receptor-activated protein function:

How can advanced structural biology techniques be applied to understand the functional mechanism of Bombesin receptor-activated protein homolog?

Applying cutting-edge structural biology techniques to understand this protein:

• Cryo-electron microscopy (cryo-EM):

  • Sample preparation strategies for membrane-associated proteins

  • Single-particle analysis for 3D structure determination

  • High-resolution structures can reveal binding pockets and interaction interfaces

  • Comparative analysis with structures of related proteins

• X-ray crystallography approach:

  • Construct design to improve crystallization propensity

  • Screening crystallization conditions with sparse matrix approach

  • Phase determination strategies for novel protein structures

  • Co-crystallization with interacting partners (e.g., ATG5)

• Integrative structural biology:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map flexible regions

  • Small-angle X-ray scattering (SAXS) for solution structure and conformational changes

  • NMR spectroscopy for dynamic regions and ligand binding studies

  • Computational modeling and molecular dynamics simulations

• AlphaFold2 and related AI methods:

  • Generate predicted structures as starting points for experimental validation

  • Compare predicted interfaces with experimental interaction data

  • Design mutations based on structure predictions for functional studies

What are the methodological considerations for investigating the role of Bombesin receptor-activated protein homolog in neuroplasticity and stress response?

Based on studies examining BC004004-/- mice in chronic unpredictable mild stress (CUMS) models:

• Behavioral testing paradigm design:

  • Forced swimming test protocol to assess depressive-like behavior

  • Sucrose preference test methodology to evaluate anhedonia

  • Open field test setup to measure anxiety-like behavior

  • Novel object recognition testing to assess memory function

• Neuronal morphology analysis:

  • Golgi staining for comprehensive dendrite visualization

  • Quantification of dendritic length and branching using Sholl analysis

  • Spine morphology classification (mature vs. immature)

  • 3D reconstruction techniques for detailed morphological analysis

• Molecular analysis of synaptic plasticity:

  • Western blotting for synaptic proteins (GluN2A, synaptophysin, BDNF)

  • Immunohistochemistry to localize expression changes

  • Electrophysiology (patch-clamp, field recordings) to assess functional changes

  • Optogenetic approaches to manipulate specific circuits

• Experimental timeline considerations:

  • Determine optimal CUMS exposure duration (21 days showed differences between genotypes)

  • Design appropriate recovery periods

  • Include multiple timepoints for analysis to capture dynamic changes

Research has shown that BC004004-/- mice are more vulnerable to stress-related disorders, exhibiting behavioral changes after 21 days of CUMS exposure when wild-type mice do not yet show such changes .

How should researchers approach comparative studies between human BRAP and Pongo abelii Bombesin receptor-activated protein C6orf89 homolog?

For effective comparative studies:

• Sequence alignment and phylogenetic analysis:

  • Perform multiple sequence alignment of BRAP homologs across species

  • Construct phylogenetic trees to understand evolutionary relationships

  • Identify conserved domains and species-specific variations

  • Calculate selection pressure (dN/dS ratios) on different protein regions

• Functional conservation testing:

  • Rescue experiments in knockout systems using orthologs from different species

  • Domain-swapping experiments to identify functionally divergent regions

  • Comparative binding assays with identified interaction partners (e.g., ATG5)

  • Cross-species cell culture models to test functional conservation in cellular context

• Expression pattern comparison:

  • Compare tissue distribution patterns between human and non-human primates

  • Analyze developmental expression profiles

  • Investigate stress-responsive expression changes across species

  • Study cell-type specific expression using single-cell RNA sequencing data

• Methodological considerations for cross-species analyses:

  • Adjust antibody selection for epitope conservation across species

  • Design primers/probes that account for sequence differences

  • Consider differences in post-translational modifications

  • Account for potential differences in protein-protein interaction networks

What experimental approaches can determine if functional differences exist between the mouse homolog (BC004004) and primate Bombesin receptor-activated proteins?

To determine functional differences between mouse and primate homologs:

• Cross-species complementation assays:

  • Express Pongo abelii or human BRAP in BC004004-/- mouse cells

  • Test rescue of phenotypes in autophagy, stress response, and inflammatory pathways

  • Create chimeric proteins with domains from different species

  • Assess differential interaction with binding partners

• Comparative biochemical characterization:

  • Side-by-side activity assays using purified recombinant proteins

  • Determine differences in post-translational modifications

  • Compare protein stability and half-life

  • Assess differential subcellular localization patterns

• Disease model comparison:

  • Test both proteins in the same experimental systems:

    • Bleomycin-induced fibrosis models

    • IMQ-induced skin inflammation

    • CUMS behavioral models

  • Measure species-specific differences in protective effects

• High-throughput approaches:

  • Comparative interactome analysis using AP-MS or BioID

  • Phosphoproteomic analysis to identify differential regulation

  • CRISPR screens to identify species-specific genetic interactions

  • Transcriptomic analysis of cells expressing homologs from different species