Bcl 2 Human (1-206 a.a.)

What is the molecular structure of human Bcl-2 (1-206 a.a.) and how does it relate to its anti-apoptotic function?

Human Bcl-2 (1-206 a.a.) is a 26 kDa protein containing four Bcl-2 homology domains (BH1-BH4) that form a hydrophobic groove capable of binding BH3 domains of pro-apoptotic proteins. The protein functions primarily by sequestering BH3-only proteins, preventing them from activating the pro-apoptotic executioner proteins BAX and BAK .

How does Bcl-2 expression change during cellular differentiation and what methodologies can detect these changes?

Bcl-2 expression is dynamically regulated during cellular differentiation, as evidenced in dendritic cells (DCs) where expression decreases during maturation. Research shows that immature CD11c+MHC-II(I-A)+ DCs express significantly higher Bcl-2 levels than mature CD11c+MHC-II(I-A)++ DCs .

To accurately detect these changes, researchers should:

• Use intracellular staining with specific anti-Bcl-2 antibodies followed by flow cytometry

• Calculate the ratio between mean fluorescence intensities (MFI) of Bcl-2 staining and isotype control staining to correct for background signals

• Include positive control populations (such as CD8+ T cells that express high Bcl-2 levels) within samples to validate staining efficacy

• Complement flow cytometry data with Western blot or RT-qPCR analysis

What distinguishes Bcl-2 from other pro-survival Bcl-2 family members in terms of binding specificity?

Bcl-2 demonstrates distinctive binding preferences among pro-survival family members (including Bcl-xL, Bcl-w, Mcl-1, Bfl-1/A1, and Bcl-B). These differences stem from subtle variations in the BH3-binding groove structure:

These binding specificities create a complex interaction network that determines cellular susceptibility to apoptosis . Understanding these distinctions is crucial for designing specific inhibitors and predicting therapeutic responses.

What are the optimal approaches for expressing and purifying recombinant human Bcl-2 (1-206 a.a.) for structural and interaction studies?

For successful expression and purification of human Bcl-2 (1-206 a.a.), researchers should consider:

• Expression system selection:

  • E. coli expression with metal affinity tags (His-tag) provides high yield but may require refolding

  • Expression in insect cells may improve solubility but with lower yields

• Optimal protocol:

  • Clone the coding sequence into a vector with an N-terminal His-tag

  • Express in E. coli BL21(DE3) strain at lower temperatures (16-18°C)

  • Purify using metal affinity chromatography followed by gel filtration

  • Consider truncating the C-terminal transmembrane domain (amino acids 207-239) to improve solubility

• Common challenges and solutions:

  • Protein aggregation: Add mild detergents (0.1% CHAPS) to purification buffers

  • Poor yield: Optimize codon usage for E. coli expression

  • Loss of function: Verify proper folding using circular dichroism and thermal shift assays

  • Stability during storage: Add 10% glycerol and store at -80°C in small aliquots

What techniques provide the most accurate analysis of Bcl-2 protein-protein interactions in research settings?

Several complementary techniques offer robust analysis of Bcl-2 protein interactions:

• Bio-layer interferometry (BLI):

  • Enables real-time measurement of association/dissociation kinetics

  • Can determine binding affinities (KD) in the nanomolar to picomolar range

  • Requires immobilization of one protein partner (typically biotinylated)

• X-ray crystallography:

  • Provides high-resolution structural data of protein complexes

  • Reveals precise binding interfaces and key interaction residues

  • Requires significant protein quantities and crystallization optimization

• Cross-linking coupled with mass spectrometry:

  • Identifies interacting regions between proteins

  • Helps validate computational design models

  • Particularly useful when crystal structures are unavailable

• Yeast surface display:

  • Allows screening of protein variants for binding specificity

  • Facilitates directed evolution approaches to enhance binding properties

  • Enables sorting of libraries under increasingly stringent conditions

• Fluorescence resonance energy transfer (FRET):

  • Enables detection of interactions in living cells

  • Provides spatial information about protein proximities

  • Requires fluorescent protein tagging that may affect native interactions

How can researchers effectively design experiments to determine the BCL2 dependency profile of cancer cell lines?

To systematically determine BCL2 dependency profiles in cancer cell lines:

• Combinatorial inhibition strategy:

  • Use highly specific inhibitors targeting individual BCL2 family members

  • Test inhibitors alone and in combinations to identify synergistic dependencies

  • Compare response patterns across multiple cell lines from the same cancer type

• Recommended methodology:

  • Treat cells with dose ranges of specific inhibitors (BH3-mimetics or designed protein inhibitors)

  • Assess viability using multiple assays (e.g., MTT, CellTiter-Glo)

  • Measure apoptosis markers (Annexin V/PI staining, caspase activation)

  • Confirm findings using genetic approaches (CRISPR knockout or RNAi)

• Data analysis approach:

  • Generate dose-response curves for single agents and combinations

  • Calculate combination indices to identify synergistic, additive, or antagonistic effects

  • Cluster cell lines based on dependency patterns

  • Correlate dependencies with baseline expression of BCL2 family members

• Validation experiments:

  • Perform BH3 profiling on permeabilized cells using synthetic BH3 peptides

  • Conduct rescue experiments by overexpressing resistant BCL2 variants

  • Analyze changes in mitochondrial membrane potential and cytochrome c release

How should researchers interpret discrepancies between Bcl-2 expression levels and clinical outcomes in different cancer types?

Interpreting discrepancies between Bcl-2 expression and clinical outcomes requires consideration of several factors:

• Context-dependent function:

  • In breast cancer, BCL2 is downregulated in HER2 and basal subtypes but may be high in luminal subtypes

  • High Bcl-2 expression paradoxically correlates with better prognosis in ER-positive breast cancers despite its anti-apoptotic function

• Methodological considerations:

  • Standardize scoring methods for immunohistochemistry (typically using >30% positive cells as threshold for positivity)

  • Consider heterogeneity within tumor samples

  • Distinguish between protein and mRNA levels, which may not correlate

• Integrated interpretation:

  • Analyze the entire BCL2 family expression profile rather than single members

  • Consider the balance between pro-survival and pro-apoptotic proteins

  • Examine post-translational modifications that affect protein function

• Functional validation:

  • Use specific BCL2 inhibitors to test functional dependency rather than relying solely on expression data

  • Consider combinatorial approaches to reveal compensatory mechanisms

What are the most effective experimental approaches to identify and overcome resistance mechanisms to Bcl-2 inhibitors?

To identify and overcome resistance to Bcl-2 inhibitors:

• Resistance mechanism identification:

  • Generate resistant cell lines through prolonged drug exposure

  • Perform whole-exome sequencing to identify mutations

  • Conduct RNA-seq to detect expression changes in apoptotic pathways

  • Use CRISPR screens to identify genes conferring resistance when knocked out

• Overcoming primary resistance:

  • Test combinatorial targeting of multiple BCL2 family members

  • For example, cells dependent on both Mcl-1 and Bcl-xL (common in colon cancers) require dual inhibition

  • Evaluate drug scheduling (sequential vs. concurrent administration)

• Addressing acquired resistance:

  • Develop next-generation inhibitors (e.g., sonrotoclax, lisaftoclax) that maintain activity against mutant Bcl-2

  • Explore alternative targeting strategies like PROTACs (proteolysis targeting chimeras) or ADCs (antibody-drug conjugates)

  • Target upstream regulators of BCL2 family expression

• Translational considerations:

  • Develop companion diagnostics to predict response

  • Monitor emergence of resistant clones during therapy

  • Identify biomarkers of early relapse

How does the differential expression of Bcl-2 across cancer subtypes inform therapeutic strategies?

Differential Bcl-2 expression patterns across cancer subtypes significantly impact therapeutic approaches:

• Hematologic malignancies:

  • High Bcl-2 expression in follicular lymphoma (85% with t(14;18) translocation) makes these cancers particularly sensitive to Bcl-2 specific inhibitors

  • Venetoclax has transformed treatment of several hematologic malignancies with manageable toxicity profiles

• Solid tumors:

  • Different dependencies identified through combinatorial inhibition:

    • Many colon cancers depend on Mcl-1 and Bcl-xL rather than Bcl-2

    • Some melanomas show primary dependence on Mcl-1 rather than Bfl-1, despite mRNA profiles suggesting otherwise

    • RKO cells demonstrate unique sensitivity to Bfl-1 inhibition

• Predictive biomarkers approach:

  • While mRNA profiling initially suggested similar BCL2 profiles between resistant HCT-116 and sensitive lines like Caco-2 and HT-29, functional testing revealed distinct dependencies

  • Protein-level analysis is more predictive than mRNA expression alone

• Toxicity management strategies:

  • BCL-xL inhibition causes on-target thrombocytopenia

  • MCL1 inhibition results in cardiac toxicities

  • Tumor-specific delivery approaches may overcome these limitations

What computational and structural approaches can researchers use to design highly specific inhibitors targeting Bcl-2 versus other family members?

Developing highly specific Bcl-2 inhibitors requires sophisticated computational and structural approaches:

• De novo protein design strategy:

  • Using stable protein scaffolds (e.g., BINDI) as starting points

  • Computational redesign of interaction surfaces to achieve high specificity

  • This approach has successfully generated inhibitors with 100-100,000 fold specificity for individual BCL2 family members

• Structure-guided optimization:

  • Crystal structures of inhibitor-target complexes (e.g., αMCL1- Mcl-1 at 2.75 Å resolution) provide detailed binding information

  • Analysis of binding pockets reveals subtle differences that can be exploited for specificity

  • Precise positioning of designed sidechains enables high affinity and selectivity

• Directed evolution approaches:

  • Single-site saturation mutagenesis (SSM) libraries to identify affinity-enhancing mutations

  • Combinatorial libraries of beneficial mutations

  • FACS-based screening under increasingly stringent conditions

  • Deep sequencing analysis of sorted populations to inform manual optimization

• Validation and refinement:

  • Cross-validation through binding assays against all family members

  • Structural confirmation through crystallography or cross-linking studies

  • Functional validation in cellular contexts

How can researchers distinguish between canonical apoptotic functions and non-canonical roles of Bcl-2 in experimental systems?

To distinguish between canonical and non-canonical Bcl-2 functions:

• Domain-specific mutants approach:

  • Generate Bcl-2 variants with mutations in specific domains (BH1-4)

  • BH3-binding groove mutants that cannot bind pro-apoptotic proteins but retain other functions

  • BH4 domain mutants to specifically disrupt non-canonical functions

• Subcellular localization studies:

  • Create Bcl-2 variants with specific localization signals (mitochondrial, ER, nuclear)

  • Compare phenotypic effects of differentially localized variants

  • Use microscopy and fractionation to confirm localization

• Temporal dynamics analysis:

  • Use inducible expression systems to distinguish immediate versus delayed effects

  • Implement rapid protein degradation systems (e.g., AID, dTAG) for acute depletion

  • Compare acute versus chronic inhibition phenotypes

• Interactome profiling:

  • Perform immunoprecipitation coupled with mass spectrometry under different conditions

  • Compare Bcl-2 interaction partners in apoptotic versus non-apoptotic contexts

  • Validate novel interactions using reciprocal pulldowns and proximity ligation assays

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and metabolomics data

  • Use bioinformatic approaches linking -omics with structural data

  • Develop network models to distinguish direct from indirect effects

What experimental systems best model the regulatory role of Bcl-2 in immune cell development and function?

To study Bcl-2's role in immune regulation, researchers should consider:

• Transgenic mouse models:

  • Cell-type specific Bcl-2 expression using lineage-specific promoters

  • Example: CD11c promoter-driven hBcl-2 expression in dendritic cells demonstrated that Bcl-2 controls DC longevity in vivo

  • Inducible systems allowing temporal control of Bcl-2 expression

• Flow cytometry analysis approach:

  • Multi-parameter staining to identify specific immune populations

  • Careful gating strategies to distinguish immature (CD11c+MHC-II+) from mature (CD11c+MHC-II++) DCs

  • Accurate Bcl-2 quantification using ratio of specific staining to isotype control

• Functional immunological assays:

  • T cell activation assays to assess DC function

  • Antigen presentation capacity measurements

  • Cytokine production analysis

  • Migration assays to assess trafficking capability

• In vivo immunization models:

  • Transfer of antigen-pulsed DCs to evaluate CTL activation

  • Measurement of humoral responses

  • Assessment of memory formation

• Single-cell approaches:

  • scRNA-seq to capture heterogeneity within immune populations

  • Trajectory analysis to identify developmental stages where Bcl-2 is critical

  • Spatial transcriptomics to understand Bcl-2 regulation in tissue microenvironments

What emerging technologies might overcome the current limitations in targeting Bcl-2 family proteins for cancer therapy?

Several cutting-edge approaches show promise for overcoming current limitations:

• Proteolysis targeting chimeras (PROTACs):

  • Bifunctional molecules that recruit E3 ligases to degrade Bcl-2 proteins

  • May achieve greater specificity than BH3-mimetics

  • Could potentially overcome resistance mechanisms

  • Allow for controlled degradation of target proteins

• Antibody-drug conjugates (ADCs):

  • Tumor-specific delivery of cytotoxic payloads

  • Reduces off-target toxicities in normal tissues

  • May overcome the thrombocytopenia limitation of BCL-xL inhibitors

  • Could enable safer targeting of MCL1 without cardiac toxicities

• BH4 domain-targeting approaches:

  • The BH4 domain is less conserved than the BH3-binding groove

  • May provide greater selectivity between family members

  • Could potentially disrupt non-canonical functions

• Spatiotemporally controlled targeting:

  • Tissue-specific and inducible promoter systems

  • Allows investigation of biological roles with high spatiotemporal control

  • Overcomes limitations of systemic small molecule distribution

  • More specific than complete protein elimination via CRISPR or RNAi

• RNA-targeting therapies:

  • Antisense oligonucleotides

  • siRNA delivery systems

  • mRNA destabilizing approaches

How can researchers develop more comprehensive experimental models to understand the complex interplay between different Bcl-2 family members?

To better understand Bcl-2 family interplay:

• Multiplexed genetic manipulation:

  • CRISPR-based screens targeting multiple family members simultaneously

  • Inducible expression systems with orthogonal control

  • Base editing to introduce specific mutations rather than knockouts

• Proximity-based interaction mapping:

  • BioID or APEX2 proximity labeling to identify spatial interaction networks

  • Split-protein complementation assays to visualize interactions in living cells

  • Advanced FRET/BRET systems with improved dynamic range

• Dynamic measurement systems:

  • Live-cell reporters of apoptotic pathway activation

  • Optogenetic control of individual protein activities

  • Biosensors to monitor conformational changes in real-time

• Patient-derived experimental models:

  • Organoids from primary tumors

  • Patient-derived xenografts

  • Ex vivo culture systems for primary cells

• Systems biology approaches:

  • Mathematical modeling of apoptotic networks

  • Machine learning algorithms to predict combinatorial effects

  • Integration of multi-omics data to build comprehensive regulatory maps

What are the methodological challenges in studying post-translational modifications of Bcl-2 and their impact on protein function?

Key methodological challenges and approaches include:

• Site-specific modification detection:

  • Develop specific antibodies against common Bcl-2 modifications (phosphorylation, ubiquitination)

  • Employ targeted mass spectrometry approaches (multiple reaction monitoring)

  • Use biochemical enrichment strategies for low-abundance modified forms

• Modification dynamics analysis:

  • Pulse-chase approaches to determine modification turnover rates

  • Single-cell techniques to capture cell-to-cell variability

  • Time-resolved proteomics following cellular stimulation

• Functional impact assessment:

  • Generate modification-mimetic mutants (e.g., phosphomimetic S→D/E substitutions)

  • Create modification-resistant mutants (e.g., S→A substitutions)

  • Develop conformation-specific antibodies that detect functionally distinct states

• Structural consequences:

  • Use hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • NMR studies of modified versus unmodified proteins

  • Molecular dynamics simulations to predict modification effects

• Spatiotemporal regulation:

  • Develop biosensors to track modifications in live cells

  • High-resolution microscopy to detect subcellular localization changes

  • Correlate modifications with protein-protein interaction dynamics