Bcl 2 Human (minus BH2 domain)

What is the structural and functional significance of the BH2 domain in Bcl-2 protein?

The BH2 domain represents one of four conserved BCL-2 homology (BH) domains in the Bcl-2 protein family. Structurally, these domains together form eight α-helical fragments linked to create the functional protein . The BH2 domain (residues 187-202) plays a critical role in maintaining Bcl-2's anti-apoptotic function. Unlike the BH4 domain, which is unique to anti-apoptotic proteins, the BH2 domain is present in both anti-apoptotic proteins (like Bcl-2 and Bcl-XL) and pro-apoptotic proteins (like BAX and BAK), but not in BH3-only proteins .

The BH2 domain contributes significantly to:

• Dimer formation with pro-apoptotic proteins

• Maintenance of proper protein folding

• Stabilization of interactions with other regulatory proteins

• Mitochondrial membrane targeting and association

Research indicates that the BH2 domain works in concert with BH1 and BH3 domains to form a hydrophobic groove that can bind the BH3 domains of pro-apoptotic proteins, thereby neutralizing their function .

How does deletion of the BH2 domain alter Bcl-2's apoptotic regulation capabilities?

Deletion of the BH2 domain (residues 187-202) fundamentally alters Bcl-2's functional properties in apoptosis regulation. When the BH2 domain is removed, the protein undergoes significant changes:

• Reduced anti-apoptotic capacity: The BH2 domain is essential for the protein's complete anti-apoptotic function, and its removal compromises this protective effect .

• Altered binding profile: Bcl-2 minus BH2 domain exhibits modified interaction patterns with pro-apoptotic proteins like BAX and BH3-only proteins, potentially reducing its ability to sequester these death-promoting factors .

• Structural reconfiguration: The three-dimensional structure of the protein changes, affecting the hydrophobic groove formed by BH1, BH2, and BH3 domains that normally binds and neutralizes pro-apoptotic BH3 domains .

• Potential function reversal: Similar to what occurs with BH4 deletion, the absence of the BH2 domain might not only abrogate anti-apoptotic function but potentially convert Bcl-2 into a pro-apoptotic protein under certain conditions .

The BH2-deleted variant provides researchers with a valuable tool to dissect domain-specific functions and binding interactions in apoptotic pathways.

What are optimal experimental conditions for working with recombinant Bcl-2 Human (minus BH2 domain)?

When working with recombinant Bcl-2 Human protein lacking the BH2 domain, several experimental conditions must be carefully controlled to maintain protein stability and activity:

• Reconstitution protocol:

  • Suspend lyophilized protein in 100μl of 0.5M acetic acid

  • Incubate overnight at 4°C

  • Dilute 10-fold into the desired buffer system for experimentation

• Redox considerations:

  • Bcl-2 has a tendency to form intramolecular disulfide bonds

  • Include 5mM DTT in assay buffers to maintain proper protein conformation

  • When running SDS-PAGE analysis, increase DTT concentration to 10mM

• Storage conditions:

  • Store lyophilized protein desiccated below -18°C for long-term stability

  • After reconstitution, store at 4°C for short-term use (2-7 days)

  • For extended storage after reconstitution, keep below -18°C

  • Add carrier protein (0.1% HSA or BSA) for improved stability during storage

  • Avoid repeated freeze-thaw cycles

• Buffer composition:

  • The recombinant protein typically contains 10mM Tris-HCL pH-8, 1mM EDTA, and 250mM NaCl

  • Consider buffer compatibility when designing binding or functional assays

Adhering to these experimental conditions will help ensure consistent and reliable results when working with this specialized Bcl-2 variant.

How can Bcl-2 (minus BH2 domain) be used as a control in apoptosis research?

Bcl-2 Human (minus BH2 domain) serves as a valuable control in multiple apoptosis research applications:

• Protein-protein interaction studies:

  • As a negative control in co-immunoprecipitation experiments to demonstrate domain-specific binding

  • In competitive binding assays to determine the contribution of the BH2 domain to interaction affinity

  • For mapping interaction surfaces with BH3-only proteins

• Functional assays:

  • As a comparison to wild-type Bcl-2 in cell survival assays

  • In mitochondrial permeabilization experiments to assess domain requirements

  • For cytochrome c release assays to evaluate anti-apoptotic capacity

• Western blotting applications:

  • As an input marker or positive control when studying domain-specific antibodies

  • For size comparison with wild-type or other domain deletion mutants

• Drug development research:

  • As a control to evaluate domain-specific requirements for drug binding

  • In resistance mechanism studies for Bcl-2 inhibitors like venetoclax

  • For screening assays to identify compounds that target other functional domains

The key advantage of using this protein variant lies in its ability to isolate the functional contribution of the BH2 domain while maintaining the integrity of other domains, allowing researchers to parse out domain-specific effects in complex biological systems.

How does the absence of the BH2 domain affect Bcl-2's interaction network in apoptotic signaling cascades?

The removal of the BH2 domain creates significant alterations in Bcl-2's interaction network within apoptotic signaling pathways. Advanced research has revealed several critical insights:

The BH2 domain functions cooperatively with BH1 and BH3 domains to form a hydrophobic pocket that normally binds and sequesters the BH3 domains of pro-apoptotic proteins . Without the BH2 domain, this binding pocket is disrupted, causing:

• Reduced binding affinity for pro-apoptotic proteins: The interaction with BAX and BAK is compromised, limiting Bcl-2's ability to prevent these proteins from forming oligomers that permeabilize the mitochondrial membrane .

• Altered selectivity profile: While wild-type Bcl-2 binds various BH3-only proteins with different affinities, BH2 domain deletion can fundamentally change this selectivity pattern .

• Disrupted higher-order protein complexes: The BH2 domain contributes to conformational stability that impacts formation of multi-protein regulatory complexes at the mitochondrial membrane .

• Modified regulatory feedback loops: Bcl-2 minus BH2 domain may alter signaling networks connecting apoptosis to other cellular processes like autophagy, as Bcl-2 normally suppresses autophagy by binding Beclin (ATG7) .

Experimental approaches to characterize these altered interaction networks include:

• Isothermal titration calorimetry to quantify binding affinities

• Proximity ligation assays to visualize protein interactions in situ

• Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• FRET-based assays to monitor real-time interactions in living cells

Understanding these modified interaction patterns provides valuable insights into domain-specific functions and may inform therapeutic strategies targeting specific interactions.

What are the implications of BH2 domain deletion for Bcl-2's role in autophagy regulation?

The deletion of the BH2 domain has significant implications for Bcl-2's role in autophagy regulation, representing an important area of investigation at the intersection of cell death and survival pathways:

• Altered Beclin-1 binding: Wild-type Bcl-2 suppresses autophagy by binding Beclin-1 (ATG7), an essential component of the mammalian autophagy system . The BH2 domain contributes to this interaction, and its deletion may compromise Bcl-2's ability to regulate autophagy, potentially creating a protein that maintains some anti-apoptotic function while losing autophagy inhibition.

• Subcellular localization effects: The anti-autophagic function of Bcl-2 appears to be primarily mediated from the endoplasmic reticulum (ER) rather than mitochondria . The BH2 domain plays a role in proper ER localization, and its deletion may alter Bcl-2 distribution between cellular compartments.

• Functional uncoupling: BH2 domain deletion offers an opportunity to potentially uncouple Bcl-2's anti-apoptotic and anti-autophagic functions, providing a valuable research tool to study the relationship between these processes.

Methodological approaches to investigate these implications include:

• Autophagosome formation assays using LC3-GFP

• Beclin-1 binding assays comparing wild-type and BH2-deleted Bcl-2

• Subcellular fractionation studies to assess compartment-specific localization

• Functional autophagy flux measurements using tandem mRFP-GFP-LC3 constructs

• Comparative studies in nutrient deprivation conditions to assess autophagy regulation

This research area has particular significance for cancer therapy, as both apoptosis evasion and autophagy dysregulation contribute to treatment resistance in many malignancies.

How can Bcl-2 (minus BH2 domain) be used to study mechanisms of resistance to Bcl-2 inhibitors?

Bcl-2 (minus BH2 domain) represents a valuable experimental tool for investigating resistance mechanisms to Bcl-2 inhibitors like venetoclax. Several methodological approaches leverage this variant:

• Binding site investigation: The BH2 domain contributes to the binding pocket targeted by small molecule inhibitors. Using the BH2-deleted variant alongside wild-type Bcl-2 in biochemical assays can help map inhibitor binding sites and identify alternative binding modes that might emerge in resistance scenarios .

• Mutation analysis comparison: Specific mutations in Bcl-2, such as F104L and F104C, have been identified as venetoclax-resistance mutations that reduce binding affinity without altering interactions with pro-apoptotic proteins . Introducing these mutations in both wild-type and BH2-deleted backgrounds can reveal how domain context influences resistance-conferring mutations.

• Structure-function studies: Comparing crystal structures of inhibitor binding to wild-type versus BH2-deleted Bcl-2 can identify conformational changes that impact drug efficacy and suggest strategies to overcome resistance.

• Cellular resistance model development:

  • Express BH2-deleted Bcl-2 in cancer cell lines

  • Compare venetoclax sensitivity to wild-type Bcl-2-expressing cells

  • Monitor changes in apoptotic threshold

  • Evaluate combinatorial treatment approaches

• BH3 profiling: This technique measures mitochondrial response to BH3 peptides and can be used to compare how BH2 deletion affects cellular dependence on Bcl-2 anti-apoptotic function and predict response to Bcl-2 inhibitors .

The knowledge gained from these approaches can inform the development of next-generation inhibitors capable of overcoming resistance or identify synergistic drug combinations for clinical use.

What methodological approaches can quantify the structural changes in Bcl-2 upon BH2 domain deletion?

Several sophisticated biophysical and structural biology techniques can be employed to quantify the structural changes in Bcl-2 when the BH2 domain is deleted:

• X-ray crystallography:

  • Provides high-resolution structural data

  • Can directly visualize conformational changes caused by domain deletion

  • Enables comparison of ligand binding pockets between wild-type and mutant protein

  • Challenges include obtaining diffraction-quality crystals of the deletion variant

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Allows analysis of protein dynamics in solution

  • Can map changes in chemical environment of specific residues

  • HSQC spectra provide fingerprints of structural perturbations

  • Especially valuable for identifying long-range conformational effects of domain deletion

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Measures solvent accessibility changes in protein regions

  • Can identify regions that become more exposed or protected after BH2 deletion

  • Provides insights into dynamic structural changes without requiring protein crystals

  • Particularly useful for mapping effects on the hydrophobic groove formed by BH1, BH2, and BH3 domains

• Circular Dichroism (CD) spectroscopy:

  • Quantifies changes in secondary structure content

  • Can detect major structural reorganizations

  • Enables thermal stability comparisons between wild-type and deletion variants

• Small-Angle X-ray Scattering (SAXS):

  • Provides low-resolution envelope of protein in solution

  • Can detect global conformational changes and protein flexibility

  • Complements high-resolution structural techniques

These approaches, used in combination, can comprehensively characterize how BH2 domain deletion impacts the three-dimensional structure and dynamics of Bcl-2, providing insights into structure-function relationships critical for understanding domain-specific roles in apoptosis regulation.

What functional assays can effectively measure the anti-apoptotic activity of Bcl-2 (minus BH2 domain)?

To effectively measure and compare the anti-apoptotic activity of Bcl-2 (minus BH2 domain) with wild-type Bcl-2, researchers can employ several complementary functional assays:

• Cell viability assays following apoptotic stimuli:

  • MTT or resazurin-based metabolic assays

  • Flow cytometry with Annexin V/PI staining to quantify early and late apoptotic populations

  • Real-time monitoring of caspase activation using fluorescent reporters

  • Long-term clonogenic survival assays to measure reproductive cell death

• Mitochondrial function assays:

  • Cytochrome c release assays from isolated mitochondria

  • JC-1 or TMRE staining to measure mitochondrial membrane potential

  • Calcium retention capacity measurements of isolated mitochondria

  • BAX/BAK oligomerization detection using crosslinking approaches

• In vitro biochemical assays:

  • Liposome permeabilization assays with reconstituted systems

  • FRET-based assays measuring BH3 domain displacement from Bcl-2

  • Co-immunoprecipitation of Bcl-2 variants with pro-apoptotic partners

  • Surface plasmon resonance to measure binding kinetics with partner proteins

• BH3 profiling technique:

  • Exposes mitochondria to titrated amounts of BH3 peptides

  • Measures mitochondrial outer membrane permeabilization

  • Compares "priming" state between cells expressing wild-type versus BH2-deleted Bcl-2

  • Provides predictive information about cellular dependency on specific anti-apoptotic proteins

These methodological approaches should be conducted under standardized conditions, and researchers should be mindful of the recombinant protein's storage and handling requirements to ensure reliable results .

What are the key considerations for experimental design when studying Bcl-2 (minus BH2 domain) in cancer models?

When designing experiments to study Bcl-2 (minus BH2 domain) in cancer models, several critical considerations must be addressed:

• Expression system selection:

  • Inducible expression systems prevent selection against potentially toxic proteins

  • Viral vectors with titratable expression allow dose-dependent studies

  • CRISPR-based knock-in approaches maintain endogenous regulation

  • Consider tagged versions for detection while verifying tag doesn't interfere with function

• Appropriate control groups:

  • Wild-type Bcl-2 expressing cells as primary comparison

  • Empty vector controls to account for vector effects

  • Other domain deletion variants (e.g., BH4-deleted) for domain-specific comparisons

  • Cells expressing known Bcl-2 mutants associated with cancer (e.g., F104L/C)

• Cell line selection:

  • Use cells with defined Bcl-2 family protein expression profiles

  • Consider BH3 profiling to determine baseline dependency on anti-apoptotic proteins

  • Include both hematologic and solid tumor models given Bcl-2's prominence in blood cancers

  • Test in cell lines with varying p53 status given downstream effects on apoptotic pathways

• Experimental endpoints and readouts:

  • Measure both short-term and long-term survival metrics

  • Assess effects on proliferation separate from apoptosis

  • Evaluate impact on response to standard chemotherapeutics

  • Consider effects on cancer stem cell populations

• In vivo considerations:

  • Xenograft models to assess tumor growth kinetics

  • Patient-derived xenografts for clinical relevance

  • Genetically engineered mouse models for studying in immunocompetent background

  • Pharmacological combination studies with Bcl-2 inhibitors like venetoclax

These design considerations help isolate the specific contributions of the BH2 domain to Bcl-2's oncogenic functions and may reveal new therapeutic vulnerabilities in cancers dependent on Bcl-2 for survival.

How can protein-protein interaction studies with Bcl-2 (minus BH2 domain) inform new therapeutic approaches?

Protein-protein interaction studies utilizing Bcl-2 (minus BH2 domain) can provide critical insights that inform novel therapeutic strategies:

• Mapping domain-specific interaction surfaces:

  • Comparative binding studies between wild-type and BH2-deleted Bcl-2 identify domain-specific binding interfaces

  • Alanine scanning mutagenesis combined with binding assays can pinpoint critical residues

  • Competitive binding assays can determine if different BH3-only proteins share overlapping binding sites

  • These approaches can identify new druggable pockets distinct from those targeted by existing inhibitors

• Understanding resistance mechanisms:

  • Determine how BH2 domain contributes to binding of clinical Bcl-2 inhibitors like venetoclax

  • Investigate if BH2 deletion mimics any resistance-conferring mutations identified in patient samples

  • Compare binding profiles of resistant cell lines to those expressing BH2-deleted Bcl-2

  • This information can guide development of inhibitors that maintain activity against resistant variants

• Identifying selective vulnerability windows:

  • Specific interaction changes in BH2-deleted Bcl-2 may reveal conditional dependencies

  • Synthetic lethality screens can identify genes/pathways that become essential when BH2 function is compromised

  • These dependencies could represent therapeutic targets in tumors with altered Bcl-2 function

• Enabling combination therapy rational design:

  • Understanding which protein interactions remain intact in BH2-deleted Bcl-2 helps identify complementary targets

  • Interactions uniquely disrupted by BH2 deletion suggest potential synergistic drug combinations

  • BH3 profiling of cells expressing BH2-deleted Bcl-2 can predict response to various apoptosis-inducing agents

• Methodological approaches include:

  • Co-immunoprecipitation with quantitative mass spectrometry

  • Surface plasmon resonance for kinetic binding parameters

  • Proximity ligation assays for in situ interaction visualization

  • FRET/BRET-based biosensors for real-time interaction monitoring

These studies can ultimately inform the development of next-generation therapeutics with improved efficacy against resistant cancers and reduced toxicity through more selective targeting.

Comparison of Domains and Functions in Bcl-2 Family Proteins

Protocol for Handling Recombinant Bcl-2 (minus BH2 domain)

How does Bcl-2 (minus BH2 domain) advance our understanding of cancer mechanisms?

The study of Bcl-2 (minus BH2 domain) provides unique insights into cancer biology and treatment resistance mechanisms:

By systematically comparing wild-type and BH2-deleted Bcl-2 across various cancer models and treatment conditions, researchers can develop more targeted therapeutic approaches that overcome resistance mechanisms and improve patient outcomes.

What therapeutic strategies can be developed based on understanding Bcl-2 domain functions?

Understanding the domain-specific functions of Bcl-2, particularly through studies of the BH2-deleted variant, informs several promising therapeutic approaches:

• Domain-specific inhibitor development:

  • Current Bcl-2 inhibitors like venetoclax primarily target the hydrophobic groove formed by multiple domains

  • Domain-specific inhibitors might overcome certain resistance mechanisms

  • Compounds targeting domains other than BH2 could maintain efficacy against BH2 domain mutations

• Combination therapy rational design:

  • BH2 domain deletion may sensitize cells to specific apoptosis inducers

  • Identifying these vulnerabilities suggests rational drug combinations

  • For example, if BH2 deletion primarily affects interactions with specific BH3-only proteins, drugs that induce those proteins might show synergy

• Antisense oligonucleotide approaches:

  • Antisense therapies targeting Bcl-2 have shown promise in increasing chemotherapy sensitivity

  • Domain-specific antisense strategies could be developed based on understanding which domains are most critical in specific cancers

  • The therapeutic principle involves introducing oligonucleotides complementary to target mRNA sequences, forming heteroduplexes that are destroyed by RNase H

• BH3 mimetic optimization:

  • BH3 mimetics mimic the action of pro-apoptotic BH3-only proteins

  • Understanding how BH2 deletion affects binding of different BH3 domains can guide development of more selective mimetics

  • Hydrocarbon-labeled peptides targeting specific binding interactions represent a promising approach

• Addressing autophagy connections:

  • Bcl-2 suppresses autophagy by binding Beclin (ATG7)

  • If BH2 deletion differentially affects autophagy regulation versus apoptosis inhibition, this could be exploited therapeutically

  • Dual-targeting of apoptosis and autophagy pathways might overcome resistance mechanisms

These therapeutic strategies represent significant advances beyond conventional cytotoxic chemotherapy, offering more targeted approaches with potentially reduced side effects and improved efficacy against resistant disease.

What are the future research directions for studying Bcl-2 domain functions?

Future research on Bcl-2 domain functions, particularly focusing on the BH2 domain, holds significant promise for advancing both basic science understanding and clinical applications:

• Comprehensive domain interactome mapping:

  • Systematic comparison of protein interaction networks between wild-type and domain-deleted variants

  • Application of proximity labeling techniques to identify transient or weak interactions

  • Development of domain-specific interaction maps across different cellular contexts and stress conditions

• High-resolution structural studies:

  • Cryo-electron microscopy of Bcl-2 family protein complexes with and without the BH2 domain

  • Time-resolved structural studies to capture conformational dynamics during apoptotic signaling

  • Structural characterization of domain-deleted variants bound to various partners and inhibitors

• Systems biology approaches:

  • Network analysis of how domain deletions impact broader signaling networks

  • Mathematical modeling of apoptotic thresholds with domain-specific perturbations

  • Machine learning to predict functional consequences of domain mutations in patient samples

• Integrated multi-omics analysis:

  • Correlation of domain-specific functions with transcriptomic, proteomic, and metabolomic changes

  • Identification of biomarkers that predict dependency on specific domains

  • Discovery of synthetic lethal interactions with domain-specific functions

• Translational and clinical applications:

  • Development of domain-function biomarkers to guide therapy selection

  • Design of domain-specific inhibitors with reduced resistance potential

  • Clinical trials incorporating domain function assessment in patient stratification

• Novel therapeutic modalities:

  • Protein degradation approaches (PROTACs) targeting specific Bcl-2 conformations

  • Domain-specific antibody or nanobody development

  • Gene editing strategies to modify specific domains rather than complete gene knockout

These research directions will deepen our understanding of how the modular architecture of Bcl-2 contributes to its function in health and disease, ultimately leading to more effective therapeutic strategies for cancer and other disorders of apoptotic regulation.