PEBP1 Human

What is human PEBP1 and what are its primary functions in cellular signaling?

PEBP1 is a highly conserved protein that functions as a key regulator of multiple signaling pathways in human cells. Structurally, it contains a remarkably conserved ligand-binding pocket that can accommodate various molecules including phospholipids and nucleotides.

PEBP1 primarily regulates three key mammalian signaling pathways:

• The Raf/MEK/ERK pathway, where it acts as an inhibitor by binding to Raf-1

• The NF-κB pathway, by binding to NF-κB inducing kinase (NIK)

• G-protein coupled receptor (GPCR) signaling, through inhibition of G-protein-coupled receptor kinase 2 (GRK2)

These interactions result in PEBP1's involvement in cellular proliferation, differentiation, migration, survival, and apoptosis . The binding of PEBP1 to these protein kinases leads to their inhibition, as demonstrated with Raf-1 where PEBP1 binding prevents phosphorylation required for Raf-1 activity .

How does PEBP1 structure relate to its binding properties?

Human PEBP1 has a well-defined three-dimensional structure with a conserved ligand-binding pocket that serves as the primary binding site for multiple ligands. X-ray crystallography studies have revealed that this pocket can accommodate various ions and small molecules such as acetate, phosphorylethanolamine (PE), phosphate, phosphotyrosine, and cacodylate .

The binding pocket is formed by 16 specific residues at the protein surface: D70, A73, P74, Y81, W84, H86, V107, G108, G110, P111, P112, H118, Y120, L180, Y181, and L184 . These residues create a versatile binding environment that allows PEBP1 to interact with diverse ligands.

NMR studies under near-physiological conditions have demonstrated that this conserved pocket can bind various phospholipids including DHPE, DHPS, DHPG, and DHPA . Additionally, nucleotide binding studies have shown affinity for molecules like FMN, GTP, GDP, and others, with binding affinities varying in a specific order (FMN>GTP>GDP>GMP>FAD>ATP) .

What techniques are most effective for studying human PEBP1-protein interactions?

Several complementary techniques have proven effective for studying PEBP1-protein interactions:

• NMR Spectroscopy: 15N-1H heteronuclear single quantum coherence (HSQC) NMR experiments can identify residues involved in binding interactions. This approach has successfully mapped binding sites for various ligands including GTP, FMN, and Raf-1 peptides. The chemical shift changes observed in HSQC spectra provide detailed information about the binding interface .

• Mass Spectrometry-Based Methods:

  • Native mass spectrometry can determine affinity constants (KD) for different ligands

  • Mass spectrometry-based cellular thermal shift assay (MS-CETSA) can detect changes in protein stability upon binding, as demonstrated in studies of PEBP1's role in mitochondrial stress response

• Luminescence Complementation Assays: Real-time interaction analysis using luminescence complementation in live cells has revealed interactions between PEBP1 and binding partners such as eIF2α .

• Co-immunoprecipitation: This technique has been used to verify protein-protein interactions involving PEBP1 in various signaling pathways.

When studying Raf-1 binding specifically, researchers should note that the minimal region of Raf-1 required for binding corresponds to amino acids 331-349, and that phosphorylation status affects binding properties .

How does PEBP1 function at the intersection of ferroptosis and autophagy?

PEBP1 acts as a critical rheostat between ferroptotic cell death and prosurvival autophagy, particularly in inflammatory conditions such as asthma. Methodologically, this function can be studied through several approaches:

• Complex Formation Analysis: PEBP1 forms complexes with 15-lipoxygenase-1 (15LO1), which can be detected through co-immunoprecipitation and proximity ligation assays. This complex is crucial for generating proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) .

• Lipid Peroxidation Assays: The 15LO1-PEBP1 complex generates 15-HpETE-PE, a ferroptotic phospholipid that can be measured using LC-MS/MS techniques .

• Autophagy Markers: Concurrent with ferroptotic signaling, PEBP1 influences autophagy by interacting with microtubule-associated light chain-3 (LC3). Monitoring LC3-I lipidation to LC3-II provides a measure of autophagy activation .

• Cell Death Assays: Ferroptotic death can be assessed through cell viability assays with ferroptosis inhibitors as controls.

Research has shown that under Type 2 inflammatory conditions, PEBP1 simultaneously promotes generation of ferroptotic signals while activating protective autophagy. This concurrent activation prevents excessive cell death and mitochondrial DNA release, a finding observed both in vitro and in Type 2 Hi asthmatic epithelial cells .

What is the role of PEBP1 in mitochondrial stress response pathways?

PEBP1 serves as an amplifier of mitochondrial stress signals and plays a crucial role in the integrated stress response (ISR). To study this function, researchers can employ:

• Thermal Stability Assays: MS-CETSA has revealed that PEBP1 is thermally stabilized specifically by stresses that induce mitochondrial ISR, suggesting a conformational change upon stress activation .

• Genetic Manipulation: Knockdown and knockout studies have demonstrated that PEBP1 depletion impairs mitochondrial ISR activation by reducing eIF2α phosphorylation and subsequent ISR gene expression .

• Protein-Protein Interaction Studies: PEBP1 interacts with eIF2α, which can be visualized using luminescence complementation in live cells. This interaction is disrupted when eIF2α is phosphorylated at Ser51 .

• Pathway-Specific Stress Inducers: Different ISR-inducing compounds can help distinguish PEBP1's role in various stress pathways. For example, complex I inhibitors like rotenone induce mitochondrial stress, while tunicamycin induces endoplasmic reticulum stress. PEBP1 appears specifically involved in the mitochondrial branch of ISR .

Experimental evidence indicates that PEBP1 acts independently of its known role in inhibiting the RAF/MEK/ERK pathway when amplifying mitochondrial stress signals, suggesting a distinct mechanism for this function .

How is PEBP1 implicated in human diseases?

PEBP1 has been associated with several significant human diseases:

Research methodologies to study PEBP1 in disease contexts should include tissue-specific expression analysis, functional assays relevant to the disease pathway, and correlation studies between PEBP1 levels/activity and disease severity .

What experimental approaches can resolve contradictory findings about PEBP1 function?

Contradictory findings regarding PEBP1 function can be addressed through several methodological approaches:

• Species-Specific Analysis: Studies have shown different binding behaviors between PEBP1 from different species (mouse, rat, human). Using species-matched experimental systems is critical for accurate interpretation .

• Controlled pH Conditions: PEBP1 binding properties are pH-dependent. Standardizing experimental pH conditions (ideally at physiological pH 7.4) can help resolve discrepancies .

• Domain-Specific Mutations: Creating targeted mutations in the conserved binding pocket versus other domains can distinguish between different functional roles of PEBP1.

• Context-Dependent Studies: Examining PEBP1 function in specific cellular contexts (cell type, stress conditions, etc.) can explain apparent contradictions in broader studies.

• Phosphorylation Status Analysis: PEBP1 function is regulated by phosphorylation. For example, phosphorylation at Ser153 causes PEBP1 to dissociate from Raf-1 and inhibit GRK2 instead . Monitoring phosphorylation states is essential for understanding contextual function.

When investigating PEBP1-Raf-1 interactions specifically, researchers should note that while the binding domains of Raf-1 with PEBP1 involve subdomains I and II, the phosphorylated N-region of Raf-1 (amino acids 331-349) is sufficient for binding .

What are the optimal conditions for studying human PEBP1 binding properties?

For optimal results when studying human PEBP1 binding properties, researchers should consider:

• pH Conditions: Maintain near-physiological pH (7.2-7.4) as binding behaviors change significantly with pH. Previous studies have used pH values ranging from 6.8 to 7.5, potentially contributing to varying results .

• Buffer Composition: Use physiologically relevant salt concentrations (approximately 150 mM NaCl) and include stabilizing agents that don't interfere with binding.

• Temperature Control: Conduct experiments at 25°C or 37°C, with consistent temperature throughout experiments to enable reliable comparisons.

• NMR Parameters: For NMR studies of PEBP1:

  • Use 15N-labeled recombinant protein

  • Record 15N-1H HSQC spectra with appropriate pulse sequences

  • Compare spectra with and without ligands to identify chemical shift perturbations

• Ligand Selection: When testing nucleotides, consider the established affinity order (FMN>GTP>GDP>GMP>FAD>ATP). For protein interactions, use defined peptide regions such as the Raf-1 331-349 sequence .

• Protein Quality: Ensure proper folding and activity of recombinant PEBP1 before binding studies, as misfolded protein can lead to spurious results.

How can researchers effectively investigate PEBP1's role at the intersection of different cellular pathways?

To effectively study PEBP1's role at pathway intersections, researchers should employ multifaceted approaches:

• Temporal Analysis: Capture the dynamic nature of PEBP1 interactions using time-course experiments to determine the sequence of events when PEBP1 shifts between different binding partners.

• Competitive Binding Assays: Determine how different binding partners compete for PEBP1 interaction by introducing multiple potential partners simultaneously.

• Mutational Analysis: Create mutations that selectively disrupt specific interactions while preserving others to dissect the functional importance of each interaction.

• Quantitative Proximity Assays: Use FRET, BRET, or split luciferase systems to measure protein-protein interactions in living cells under different conditions.

• Integrated Multi-Omics: Combine proteomics, lipidomics, and transcriptomics to capture the full spectrum of changes when PEBP1 function is perturbed.

For investigating PEBP1's role between ferroptosis and autophagy specifically, researchers should measure both LC3-II formation and lipid peroxidation simultaneously in the same system while manipulating PEBP1 levels . Similarly, when studying its role in mitochondrial stress response, monitoring both eIF2α phosphorylation and downstream gene expression provides a more complete picture .

What are the challenges in resolving the complete interactome of human PEBP1?

Mapping the complete interactome of human PEBP1 presents several methodological challenges:

• Transient Interactions: Many of PEBP1's interactions may be transient or context-dependent, making them difficult to capture with traditional techniques. Approaches like chemical crosslinking combined with mass spectrometry can help stabilize and identify these interactions.

• Competition Between Partners: PEBP1 interacts with multiple partners (15LO1, LC3, eIF2α, Raf-1) that may compete for binding, requiring sophisticated experimental designs to determine binding hierarchies and preferences.

• Post-Translational Modifications: PEBP1 function is regulated by phosphorylation and possibly other modifications. A comprehensive analysis requires monitoring these states simultaneously with interaction studies.

• Tissue-Specific Interactions: PEBP1 may have different interacting partners in different tissues or disease states. Tissue-specific interactome studies using primary cells or relevant disease models provide more contextually accurate information.

• Membrane Association: As a phosphatidylethanolamine-binding protein, PEBP1 interacts with membrane components, requiring specialized techniques for membrane-associated protein interactions.

To overcome these challenges, researchers should consider proximity-dependent biotinylation (BioID or TurboID) combined with mass spectrometry to capture both stable and transient interactions in living cells, along with spatial information about where these interactions occur .

What are the most promising therapeutic targets in the PEBP1 pathway for disease intervention?

Based on current research, several promising therapeutic targets exist within the PEBP1 pathway:

• 15LO1-PEBP1 Complex: This complex generates ferroptotic signals in inflammatory conditions like asthma. Disrupting this interaction could potentially reduce inflammatory damage while preserving beneficial autophagy .

• PEBP1-eIF2α Interaction: Modulating this interaction could fine-tune the integrated stress response in mitochondrial dysfunction-related diseases .

• PEBP1 Expression in Metastasis: Restoring PEBP1 expression in metastatic cancer could exploit its metastasis suppressor function. This approach would require tissue-specific delivery systems .

• Post-Translational Modifications: Targeting the phosphorylation of PEBP1 at Ser153 could modulate its switch from Raf-1 inhibition to GRK2 inhibition, potentially affecting GPCR signaling in relevant diseases .

To effectively pursue these targets, researchers should focus on developing:

• Small molecule modulators of specific PEBP1 interactions

• Peptide-based inhibitors of the 15LO1-PEBP1 complex

• Approaches to enhance PEBP1 expression in specific tissues

• Methods to modulate PEBP1 phosphorylation state

How can contradictory findings about PEBP1 function across different human tissues be reconciled?

To reconcile contradictory findings about PEBP1 function across different tissues, researchers should implement:

• Tissue-Specific Experimentation: Conduct parallel experiments in multiple tissue types using identical methodologies to directly compare PEBP1 function across tissues.

• Single-Cell Analysis: Employ single-cell techniques to determine if apparent contradictions result from different cell populations within tissues rather than tissue-specific differences.

• Interactome Mapping: Perform comprehensive interactome studies in different tissues to identify tissue-specific binding partners that may direct PEBP1 toward different functions.

• Context-Dependent Signaling: Investigate how tissue-specific signaling environments affect PEBP1 function, particularly considering:

  • Differences in pH or redox environments

  • Varying levels of competing binding partners

  • Tissue-specific post-translational modifications

• Isoform Analysis: Determine if different PEBP1 isoforms or post-translationally modified forms predominate in different tissues.

Combining these approaches with careful consideration of experimental conditions can help reconcile seemingly contradictory findings and develop a unified model of context-dependent PEBP1 function .