BCL2 Human, His

What is BCL2 and why is it important in cellular research?

BCL2 is a critical anti-apoptotic protein that functions as an integral outer mitochondrial membrane protein, blocking the apoptotic death of certain cells, particularly lymphocytes. It belongs to the BCL2 family of proteins that regulate programmed cell death through a complex network of protein-protein interactions. The importance of BCL2 stems from its central role in the regulation of apoptosis, a process fundamental to development, tissue homeostasis, and disease pathogenesis . Dysregulation of BCL2 expression, such as through chromosomal translocation to the immunoglobulin heavy chain locus, is implicated in follicular lymphoma and other malignancies . Additionally, the BCL2 pathway has emerged as a valuable therapeutic target, with drugs like venetoclax (which targets BCL2) showing clinical efficacy in conditions such as Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) .

What is the structural basis for BCL2 function?

BCL2 proteins fold into a distinctive helical bundle structure characterized by a central hydrophobic helix (α5) that serves as a scaffold for up to eight α-helices. This folding pattern brings the BCL2 homology (BH) regions into close proximity, creating the canonical BH3-binding groove where antagonist BH3 motifs bind . The protein contains four conserved domains (BH1-BH4) that contribute to its anti-apoptotic function and specificity . Most anti-apoptotic BCL2 family members, including BCL2 itself, possess C-terminal transmembrane (TM) domains that target them to intracellular membranes, particularly the mitochondrial outer membrane . The three-dimensional arrangement of these helices creates a hydrophobic pocket formed by helices 3, 4, and 5, which is crucial for the protein's ability to sequester pro-apoptotic BH3-only proteins and prevent apoptosis initiation .

How does recombinant His-tagged BCL2 differ from native BCL2?

Recombinant His-tagged BCL2 contains a 20-amino acid histidine tag at the N-terminus that facilitates purification through metal affinity chromatography techniques . This modification, while useful for isolation and detection, may affect certain protein characteristics:

• Structural considerations: The His-tag can potentially alter protein folding dynamics, though most studies indicate minimal impact on the core BCL2 structure when placed at the N-terminus

• Binding kinetics: Tag placement may subtly influence protein-protein interaction kinetics, particularly if near functional domains

• Solubility properties: The charged His-tag can increase protein solubility in aqueous buffers

• Immunogenicity in model systems: Tagged proteins may exhibit different immunological profiles in certain experimental contexts

For research requiring native-like properties, it's advisable to either use enzymatic methods to remove the His-tag following purification or validate that the tag doesn't interfere with the specific protein functions under investigation .

What are the recommended storage conditions for maintaining BCL2 protein stability?

Optimal storage conditions for BCL2 Human His-tagged protein include:

• Short-term storage (2-4 weeks): 4°C in the original buffer formulation

• Long-term storage: -20°C in aliquots to avoid freeze-thaw cycles

• Buffer composition: 20 mM Tris-HCl (pH 8.0), 2 mM DTT, and 20% glycerol

• Addition of carrier protein (0.1% HSA or BSA) for extended storage periods to prevent adsorption and improve stability

Multiple freeze-thaw cycles significantly reduce protein activity through denaturation and aggregation. Therefore, preparing single-use aliquots is strongly recommended. Studies have shown that properly stored BCL2 protein retains >90% activity for up to 6 months when maintained under these conditions with minimal freeze-thaw cycles.

What are the optimal conditions for BCL2-BH3 binding assays?

When designing BCL2-BH3 binding assays, researchers should consider:

Binding Buffer Optimization:

Methodological Approaches:

• Fluorescence Polarization (FP): Utilizes fluorescently labeled BH3 peptides to measure binding to BCL2. Most sensitive when using FITC or TAMRA-labeled peptides at 10-50 nM concentrations .

• Surface Plasmon Resonance (SPR): Provides real-time kinetic data. Optimal when BCL2 is immobilized via the His-tag to an NTA sensor chip with BH3 peptides as analytes.

• Isothermal Titration Calorimetry (ITC): Offers thermodynamic parameters of binding. Requires higher protein concentrations (10-20 μM) but provides direct measurement without labeling.

The choice of method depends on research objectives, with FP being most suitable for screening applications, SPR for detailed kinetic studies, and ITC for comprehensive thermodynamic analysis .

How can BCL2 protein be effectively used in mitochondrial-based apoptosis assays?

Mitochondrial-based assays for BCL2 function can be implemented through several approaches:

• Cytochrome c Release Assay:

  • Isolate intact mitochondria from appropriate cell types

  • Preincubate with purified BCL2-His protein (0.1-1 μM)

  • Challenge with BH3-only proteins or BH3 peptides

  • Measure cytochrome c release via western blot or ELISA

  • Include controls with mutant BCL2 proteins to verify specificity

• JC-1 Dye-Based Membrane Potential Assay:

  • This flow cytometry method measures mitochondrial membrane potential loss

  • More efficient than cytochrome c release assays for high-throughput screening

  • Can distinguish between different cell populations in heterogeneous samples

  • Has successfully differentiated BCL2 dependence in leukemic myeloblasts versus hematopoietic stem cells

• Liposome Permeabilization Assay:

  • Reconstitute purified BCL2-His into liposomes with defined lipid composition

  • Challenge with activated BAX/BAK proteins

  • Measure dye release as indicator of membrane permeabilization

  • Allows precise control of membrane composition to study lipid effects on BCL2 function

These assays should include appropriate controls including BCL2 inhibitors like ABT-199 (venetoclax) to validate BCL2-specific effects . When interpreting results, it's crucial to consider that in vitro findings may not always translate directly to cellular contexts due to the complex interplay of multiple BCL2 family members.

What approaches can be used to study BCL2 interactions with other family members?

Several complementary approaches can be employed to characterize BCL2 interactions with other family members:

In vitro Protein-Protein Interaction Methods:

• Co-immunoprecipitation (Co-IP) with His-tag pull-down:

  • Use anti-His antibodies to pull down BCL2-His

  • Detect interacting partners via western blot

  • Include appropriate negative controls (e.g., non-relevant His-tagged protein)

• Biolayer Interferometry (BLI) or Surface Plasmon Resonance (SPR):

  • Immobilize BCL2-His on NTA sensors

  • Measure real-time binding of purified interacting partners

  • Determine binding constants (KD, kon, koff)

  • Test competitive binding with BH3 peptides or small molecules

Cellular Methods:

• Proximity Ligation Assay (PLA):

  • Visualize protein interactions in situ with subcellular resolution

  • Quantify interaction frequency in different cellular compartments

  • Compatible with fixed cells and tissues

• FRET/BRET Approaches:

  • Generate fusion constructs of BCL2 and interaction partners

  • Measure energy transfer as indicator of protein proximity

  • Monitor interactions in living cells in real-time

The evolutionary conservation of BCL2 structure from sponges to humans underscores the fundamental nature of these interactions . When analyzing interaction data, researchers should consider that different BCL2 family members may have distinct binding preferences and affinities for various BH3-only proteins, reflected in their specialized biological functions .

How does BCL2 overexpression affect human embryonic stem cell survival and applications?

BCL2 overexpression significantly enhances human embryonic stem cell (hESC) survival through multiple mechanisms:

• Resistance to Dissociation-Induced Apoptosis:

  • BCL2-overexpressing hESCs show dramatically improved survival after single-cell dissociation

  • Enhanced colony formation from sorted single cells (improved from ~1% to significantly higher rates)

  • ROCK inhibitor further enhances this effect, suggesting complementary pathways

• Enhanced Embryoid Body Formation:

  • BCL2-hESCs form embryoid bodies more efficiently

  • Improved survival during the initial aggregation phase

• Reduced Serum Dependency:

  • BCL2-hESCs exhibit normal growth in serum-free conditions

  • Still require basic fibroblast growth factor (bFGF) to remain undifferentiated

  • Show increased resistance to apoptosis in minimal media (0% knockout serum replacement) compared to control hESCs

• Preservation of Pluripotency:

  • BCL2-overexpressing hESCs maintain pluripotency markers

  • Form teratomas in vivo

  • Differentiate into all three germ layers

These findings have important implications for stem cell applications, including improved generation of disease models, enhanced cell manufacturing for therapeutic applications, and more efficient genetic modification protocols. The data suggest that modulating the BCL2 pathway may be a generally applicable approach to improve stem cell survival without compromising developmental potential .

What role does BCL2 play in cancer development and how is it targeted therapeutically?

BCL2 contributes to cancer development through multiple mechanisms and has emerged as an important therapeutic target:

Oncogenic Mechanisms:

• Inhibition of Apoptosis: BCL2 prevents cell death by sequestering pro-apoptotic proteins, allowing cancer cells to evade apoptosis triggered by oncogenic stress

• Genetic Alterations: Chromosomal translocations (e.g., t(14;18) in follicular lymphoma) place BCL2 under control of the immunoglobulin heavy chain enhancer, leading to constitutive expression

• Collaborative Oncogenesis: BCL2 cooperates with other oncogenes (e.g., MYC) by blocking the apoptotic response they would normally induce

Therapeutic Targeting Strategies:

How can researchers effectively study BCL2 mutations and their impact on protein function?

To systematically investigate BCL2 mutations and their functional consequences, researchers can implement a multi-layered approach:

Mutation Analysis Strategies:

• Site-Directed Mutagenesis:

  • Generate specific BCL2-His variants based on:

    • Cancer-associated mutations from databases like COSMIC

    • Structure-guided mutations in key functional regions (BH domains, helices)

    • Evolutionary conservation analysis to identify critical residues

  • Express and purify mutant proteins using identical conditions to wild-type

• Structural Impact Assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure changes

  • Thermal shift assays to determine protein stability

  • X-ray crystallography or cryo-EM for detailed structural analysis of key mutations

Functional Characterization:

Computational Approaches:

• Molecular dynamics simulations to predict mutation effects on protein dynamics

• In silico docking studies to evaluate altered interactions with BH3 peptides or drugs

• Sequence conservation analysis across species to prioritize mutations

When interpreting mutation data, researchers should consider both loss-of-function effects (reduced anti-apoptotic activity) and gain-of-function possibilities (altered specificity, drug resistance). Additionally, some mutations may affect post-translational modifications or protein-protein interactions outside the canonical BH3-binding groove .

How can high-throughput screening be optimized for discovering BCL2 inhibitors?

Optimizing high-throughput screening (HTS) for BCL2 inhibitors requires careful assay design and validation:

Primary Screening Assays:

• Fluorescence Polarization (FP) Binding Assays:

  • Utilize FITC or TAMRA-labeled BH3 peptides (BAD-BH3 most specific for BCL2)

  • Optimize for Z' factor >0.7 with appropriate positive controls (ABT-199)

  • Screening concentration typically 10 μM with 1-5% DMSO tolerance

  • Miniaturizable to 384 or 1536-well formats

• Time-Resolved FRET (TR-FRET) Assays:

  • Lower background than standard FP assays

  • Particularly useful for compound libraries with autofluorescent molecules

  • Requires dual-labeled components (e.g., His-tagged BCL2 with anti-His-Europium antibody)

Cascade Validation Strategy:

Data Analysis Considerations:

• Implement machine learning algorithms to identify structure-activity relationships

• Cluster hits based on chemical scaffolds and mechanism

• Prioritize compounds with selectivity for BCL2 over BCL-XL (to avoid thrombocytopenia)

• Consider physicochemical properties early to avoid developing potent but non-drug-like molecules

BCL2 inhibitor screening programs should consider the structural knowledge that the BH3-binding groove requires compounds that can mimic the amphipathic α-helical BH3 domain, presenting specific challenges for medicinal chemistry optimization .

What are the current challenges in studying BCL2 evolutionary conservation and what methods overcome these?

Studying BCL2 evolutionary conservation presents several challenges that can be addressed with specialized approaches:

Major Challenges:

• Sequence Divergence: Despite functional conservation, BCL2 family proteins show considerable sequence divergence across distant phyla

• Domain Recognition: The BH domains can be difficult to identify in distant homologs using sequence analysis alone

• Structural vs. Sequence Conservation: The three-dimensional structure is more conserved than primary sequence

• Functional Redundancy: Multiple family members with overlapping functions complicate ortholog identification

Methodological Solutions:

• Structure-Based Approaches:

  • Profile-based hidden Markov models incorporating structural information

  • Threading algorithms to identify proteins with similar fold architecture

  • These approaches have successfully identified BCL2-like proteins across metazoans

• Functional Genomics:

  • Complementation assays in model systems (e.g., can putative homolog rescue BCL2 knockout phenotype?)

  • BH3-interactome mapping across species to identify functional equivalents

  • Cross-species apoptotic pathway reconstitution

• Comparative Experimental Validation:

  Species Comparison	Experimental Approach	Key Insights
  Human vs. Mouse	Knock-in studies replacing with human BCL2	Functional conservation in mammals
  Vertebrate vs. Invertebrate	Expression of invertebrate BCL2-like proteins in human cells	Conservation of core anti-apoptotic mechanism
  Metazoan vs. Non-metazoan	Structural and biochemical comparisons	Origins of the BCL2 fold

Research suggests that the anti-apoptotic BCL2-like and pro-apoptotic BH3-only family members likely arose through duplication and modification of genes for pro-apoptotic multi-BH domain proteins like BAX and BAK . The canonical BCL2 fold that brings BH regions into proximity to form the BH3-binding groove is maintained from sponges to humans, indicating strong selective pressure on this structural feature . This evolutionary insight helps explain why targeting this conserved pocket has proven effective for developing BCL2 inhibitors with clinical utility .

How do post-translational modifications affect BCL2 function and how can these be studied?

Post-translational modifications (PTMs) of BCL2 critically regulate its function, localization, and interactions. Research approaches to study these modifications include:

Major BCL2 Post-Translational Modifications:

Experimental Approaches:

• Site-Specific Mutant Generation:

  • Create phosphomimetic (S→D/E) and phosphodeficient (S→A) BCL2 mutants

  • Express in cells or purify for in vitro studies

  • Compare functional differences in apoptosis assays

• Mass Spectrometry-Based PTM Mapping:

  • Immunoprecipitate BCL2 from cells under different conditions

  • Perform LC-MS/MS analysis with PTM-enrichment strategies

  • Use SILAC or TMT labeling for quantitative comparison

• Proximity-Based Labeling:

  • Generate BCL2-BioID or BCL2-APEX2 fusion proteins

  • Identify proteins in proximity to BCL2 under different PTM states

  • Map dynamic interaction changes upon stimulation

• Real-Time PTM Sensors:

  • Develop FRET-based sensors for specific BCL2 PTMs

  • Monitor modification dynamics in living cells

  • Correlate with apoptotic events and localization changes

Studies should consider that PTMs often work in combination, creating a complex "PTM code" that fine-tunes BCL2 function in context-specific ways. For example, multi-site phosphorylation of BCL2 during mitosis differs from phosphorylation patterns induced by survival signaling. Additionally, PTMs may differently affect BCL2's interactions with distinct BH3-only proteins, adding another layer of regulatory complexity .

What are the most promising future directions in BCL2 research?

The field of BCL2 research continues to evolve rapidly, with several promising directions for future investigation:

• Structural Biology Advances:

  • Cryo-EM studies of full-length BCL2 in membrane environments

  • Visualization of complete BCL2 interactomes in native contexts

  • Real-time conformational changes during apoptosis regulation

• Systems Biology Approaches:

  • Quantitative models of BCL2 family interaction networks

  • Single-cell analysis of BCL2 dependency across tissues

  • Integration of BCL2 signaling with other cellular pathways

• Therapeutic Innovations:

  • Development of selective MCL1 inhibitors with favorable therapeutic indices

  • Rational combination strategies with BCL2 inhibitors

  • Novel delivery systems to enhance tissue specificity

  • Biomarker development to better predict clinical responses

• Non-Apoptotic Functions:

  • Deeper exploration of BCL2's role in metabolism

  • Understanding BCL2 involvement in autophagy regulation

  • Investigation of BCL2 in cellular calcium homeostasis

• Translational Applications:

  • Engineering BCL2-overexpressing cells for enhanced survival in cell therapy applications

  • Targeting BCL2 in neurodegenerative diseases where inappropriate apoptosis occurs

  • Exploiting BCL2 biology for improved organ preservation techniques