Bcl 2 Human (minus NWGR domain)

What is the NWGR domain in human Bcl-2 and what is its molecular function?

The NWGR domain (asparagine-tryptophan-glycine-arginine) is a highly conserved motif located within the BH1 (Bcl-2 homology 1) domain of Bcl-2 protein. This tetrapeptide sequence (residues 143-146) plays a critical role in the anti-apoptotic function of Bcl-2. The NWGR motif is integral to Bcl-2's ability to:

• Mediate protein-protein interactions with pro-apoptotic family members

• Contribute to the structural stability of the hydrophobic binding groove that accommodates BH3 domains of pro-apoptotic proteins

• Participate in regulating mitochondrial membrane permeability

The importance of this domain is highlighted when comparing Bcl-2 family proteins. While anti-apoptotic members like Bcl-2 contain the NWGR sequence, some pro-apoptotic members like Bok have variations (such as TWGK), which correlates with their different functions in apoptosis regulation .

How does the removal of the NWGR domain affect the anti-apoptotic function of Bcl-2?

The deletion of the NWGR domain (residues 143-146) from human Bcl-2 significantly impacts its anti-apoptotic function through several mechanisms:

• Altered binding capacity: Without the NWGR domain, Bcl-2's ability to interact with pro-apoptotic proteins becomes compromised, weakening its capacity to neutralize these proteins.

• Structural implications: The removal disrupts the integrity of the hydrophobic groove, which is critical for sequestering BH3 domains of pro-apoptotic proteins like Bax and Bak.

• Functional consequences: Bcl-2 minus NWGR domain exhibits reduced ability to block apoptosis, as its primary mechanism of preventing mitochondrial outer membrane permeabilization is impaired.

This domain is critical for Bcl-2's role in regulating cell death by controlling mitochondrial membrane permeability and inhibiting caspase activity . Research applications frequently use this truncated form to study structure-function relationships in the Bcl-2 family.

What are the optimal methods for expressing and purifying human Bcl-2 without the NWGR domain?

Based on established protocols, the following methodological approach is recommended for optimal expression and purification of human Bcl-2 minus NWGR domain:

Expression System:

• E. coli is the preferred expression system, as demonstrated in commercial preparations

• Expression as a His-tagged fusion protein facilitates purification

• BL21(DE3) strain with IPTG induction (0.5-1.0 mM) at lower temperatures (18-25°C) improves solubility

Purification Protocol:

• Cell Lysis: Sonication in buffer containing 10mM Tris-HCl pH 8.0, 1mM EDTA, 250mM NaCl

• Initial Solubilization: Suspend lyophilized protein in 100μl of 0.5M Acetic acid, overnight at 4°C

• Buffer Exchange: Dilute 10-fold into selected buffer system

• Reducing Conditions: Include 5mM DTT in assay buffer to prevent intramolecular disulfide bond formation

• Chromatography Sequence:

  • Nickel affinity chromatography (for His-tagged constructs)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

Storage Considerations:

• Store lyophilized protein desiccated below -18°C

• Store reconstituted protein at 4°C for short-term (2-7 days)

• For long-term storage, add carrier protein (0.1% HSA or BSA) and store below -18°C

• Avoid repeated freeze-thaw cycles

This methodology consistently yields >95% pure protein as determined by RP-HPLC and SDS-PAGE analysis.

How do protein interactions of Bcl-2 change when the NWGR domain is removed?

The removal of the NWGR domain significantly alters Bcl-2's interaction profile with various proteins:

The NWGR domain plays a decisive role in Bcl-2's interactions with other family members. While wild-type Bcl-2 effectively heterodimerizes with pro-apoptotic Bcl-2 proteins through interactions involving this domain, the deletion variant exhibits altered binding preferences .

This domain also appears relevant for interactions with non-Bcl-2 family proteins. Research has revealed that galectin-7 (Gal7) directly interacts with Bcl-2, and this interaction may be influenced by the NWGR domain, similar to how the Bcl-2/Gal3 interaction is affected by the vicinity of CRD and NWGR motif .

What role does the NWGR domain play in the interaction of Bcl-2 with galectin proteins?

The NWGR domain functions as a critical interaction surface for galectin binding to Bcl-2, with several important implications:

• Structural Similarity: The NWGR motif in Bcl-2 bears striking resemblance to the carbohydrate recognition domain (CRD) found in some galectins, suggesting an evolutionary relationship or convergent functional development.

• Galectin-3 Interaction: The Bcl-2/Gal3 interaction has been shown to be lactose-inhibitable, likely due to the proximity of the CRD and NWGR motif in Gal3, indicating a carbohydrate-mediated component to this interaction .

• Galectin-7 as Novel Partner: Proteomic analysis has identified galectin-7 as a previously unknown Bcl-2 interacting partner. This interaction occurs at mitochondria, where a fraction of Gal7 is constitutively localized in a Bcl-2-dependent manner .

• Functional Consequences: When the NWGR domain is removed, these interactions are likely disrupted, potentially altering the pro- or anti-apoptotic balance within cells.

Research indicates that understanding these interactions provides insight into novel regulatory mechanisms of apoptosis. For example, galectin-7 has been shown to sensitize mitochondria to apoptotic stimuli, suggesting a counterregulatory role to Bcl-2's anti-apoptotic function .

Which techniques are recommended to study interactions between Bcl-2 (minus NWGR) and other proteins?

For comprehensive characterization of interactions between Bcl-2 (minus NWGR) and its binding partners, the following methodological approaches are recommended:

In vitro Techniques:

• Co-immunoprecipitation (Co-IP):

  • Use specific antibodies against Bcl-2 minus C-terminus (such as MAB827)

  • Include appropriate controls (IgG, Bcl-2 wild-type)

  • Analyze by western blot at approximately 24 kDa

• Pull-down Assays:

  • Utilize His-tagged Bcl-2 (minus NWGR) as bait

  • Include 5mM DTT in buffers to prevent disulfide formation

  • Evaluate binding partners by mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize Bcl-2 (minus NWGR) on sensor chip

  • Measure binding kinetics (Ka, Kd) of various partners

  • Compare with wild-type Bcl-2 to quantify affinity changes

Cellular Techniques:

• Proximity Ligation Assay (PLA):

  • Visualize protein interactions in situ

  • Quantify interaction frequency and subcellular localization

• FRET/BiFC Analysis:

  • Generate fusion constructs with fluorescent proteins

  • Monitor real-time interactions in living cells

• Mitochondrial Proteomic Approach:

  • Isolate pure mitochondrial fractions

  • Combine Bcl-2 immunocapture with mass spectrometry

  • Perform gene ontology mining to identify protein networks

This multi-technique approach allows for robust validation of interaction partners and provides insights into how the NWGR domain deletion affects Bcl-2's interactome.

What are the structural characteristics of human Bcl-2 without the NWGR domain?

Human Bcl-2 without the NWGR domain (residues 143-146) exhibits several notable structural characteristics:

The structural alterations resulting from NWGR deletion provide valuable insights into the molecular basis of Bcl-2 function and its interactions with both pro-apoptotic partners and novel interactors like galectin-7 .

How can the impact of NWGR domain removal on Bcl-2's ability to regulate mitochondrial permeability be experimentally evaluated?

To experimentally assess how NWGR domain removal affects Bcl-2's regulation of mitochondrial permeability, researchers should implement the following methodological approaches:

In vitro Mitochondrial Assays:

• Cytochrome c Release Assay:

  • Isolate intact mitochondria from relevant cell types

  • Incubate with purified Bcl-2 (minus NWGR) ± pro-apoptotic proteins

  • Measure cytochrome c release by western blot or ELISA

  • Compare with wild-type Bcl-2 control

• Mitochondrial Permeability Transition Pore (MPTP) Assays:

  • Measure calcium retention capacity in isolated mitochondria

  • Monitor mitochondrial swelling spectrophotometrically

  • Assess membrane potential using potential-sensitive dyes

Cellular Models:

• Reconstitution Studies:

  • Use Bcl-2 knockout cell lines

  • Reintroduce either wild-type or NWGR-deleted Bcl-2

  • Challenge with apoptotic stimuli (e.g., staurosporine, etoposide)

  • Quantify apoptotic endpoints

• Live-Cell Mitochondrial Imaging:

  • Utilize fluorescent probes (TMRM, JC-1) for membrane potential

  • Implement time-lapse microscopy to track dynamic changes

  • Correlate with apoptotic events using multiplexed reporters

Molecular Analysis:

• BH3 Profiling:

  • Expose mitochondria to BH3 peptides

  • Compare sensitivity patterns between wild-type and mutant Bcl-2

  • Derive mechanistic insights from differential responses

• Interaction Mapping:

  • Assess binding to key regulators of mitochondrial permeability

  • Include both Bcl-2 family members and newly identified partners like galectin-7

These approaches collectively provide a comprehensive evaluation of how the NWGR domain contributes to Bcl-2's canonical function in maintaining mitochondrial integrity and preventing apoptosis.

What cellular systems are most suitable for studying the function of Bcl-2 without the NWGR domain?

The choice of cellular systems for studying Bcl-2 minus NWGR domain should be guided by specific research objectives and the biological contexts where Bcl-2 function is most relevant:

Recommended Cell Types:

• Hematological Cell Lines:

  • KG-1 (acute myelogenous leukemia): Demonstrates detectability of Bcl-2 variants by western blot

  • MCF-7 (breast cancer): Shows reliable expression of Bcl-2 and amenability to Simple Western analysis

  • Follicular lymphoma lines: Relevant due to Bcl-2 translocations in this disease

• Engineered Systems:

  • CRISPR-modified Bcl-2 knockout lines: Allow clean reconstitution experiments

  • Tet-inducible expression systems: Enable temporal control of Bcl-2 variant expression

  • Knock-in models: For physiologically relevant expression levels

• Primary Cells:

  • Granulosa cells: Exhibit natural apoptosis regulation during follicle atresia

  • Ovarian, testicular, and uterine tissues: Show high Bok expression, providing context for comparative Bcl-2 family studies

Experimental Design Considerations:

• Expression Level Control:

  • Titrate expression to physiological levels

  • Include wild-type Bcl-2 controls at matched expression

  • Monitor with validated antibodies like MAB827

• Functional Readouts:

  • Apoptosis assays (Annexin V, caspase activation)

  • Mitochondrial integrity measurements

  • Protein-protein interaction studies

• Genetic Background:

  • Consider expression of other Bcl-2 family members

  • Account for p53 status (many cancer lines are p53-deficient)

  • Evaluate the presence of galectin-7 and other Bcl-2 interactors

The most informative systems will allow direct comparison between wild-type and NWGR-deleted Bcl-2 in contexts where Bcl-2's anti-apoptotic function is clearly demonstrable and physiologically relevant.

What applications does human Bcl-2 without the NWGR domain have in cancer research?

Human Bcl-2 without the NWGR domain serves as a valuable tool in cancer research through several key applications:

Mechanistic Studies of Apoptosis Resistance:

• Provides insights into how cancer cells evade apoptosis through Bcl-2 overexpression

• Helps delineate the precise molecular requirements for Bcl-2's anti-apoptotic function

• Allows correlation between structural features and functional consequences in cancer contexts

Drug Development and Validation:

• Serves as a control to validate specificity of BH3-mimetic drugs that target the NWGR-containing binding pocket

• Enables screening of compounds that might circumvent resistance mechanisms

• Provides a platform for developing drugs targeting alternative binding sites

Biomarker Development:

• Allows generation of specific antibodies that can distinguish between functional states of Bcl-2

• Facilitates development of diagnostic tools for assessing Bcl-2 functional status in tumors

• Enables correlation studies between structural variants and clinical outcomes

Study of Novel Interactions:

• Helps identify cancer-relevant interaction partners of Bcl-2 beyond classical Bcl-2 family members

• Illuminates the significance of interactions with proteins like galectin-7 in cancer progression

• Provides insights into non-canonical functions of Bcl-2 in cancer cells

Therapeutic Resistance Mechanisms:

• Contributes to understanding how mutations or post-translational modifications near the NWGR domain might confer resistance to Bcl-2 inhibitors

• Facilitates development of combination strategies to overcome resistance

The research applications of Bcl-2 minus NWGR domain extend beyond basic science to translational cancer research, potentially informing next-generation targeted therapies and diagnostic approaches.

How does the removal of the NWGR domain affect Bcl-2 dimerization?

The removal of the NWGR domain (residues 143-146) significantly impacts Bcl-2 dimerization properties through multiple mechanisms:

Effects on Homodimerization:

• Structural Destabilization: The NWGR motif contributes to the stability of the hydrophobic groove, and its removal likely compromises the structural integrity needed for homodimerization.

• Disulfide Bond Formation: Bcl-2 minus NWGR domain has a greater tendency to form intramolecular disulfide bonds , potentially affecting the conformation required for proper dimerization.

• Technical Implications: When working with this variant, 5mM DTT is recommended in assay buffers and 10mM DTT for SDS-PAGE to prevent inappropriate disulfide formation .

Effects on Heterodimerization:

• Pro-apoptotic Partner Binding: The NWGR motif is critical for binding BH3 domains of pro-apoptotic proteins. Its removal would significantly reduce heterodimerization with proteins like Bax and Bak.

• Selectivity Changes: Similar to how Bok (with TWGK instead of NWGR) shows selective binding to only certain anti-apoptotic members (Mcl-1, BHRF1, and Bfl-1) , Bcl-2 minus NWGR likely exhibits altered binding selectivity.

• Non-Bcl-2 Family Interactions: The domain removal may also affect interactions with non-canonical partners like galectin-7, which has been identified as a novel Bcl-2 binding protein through proteomic approaches .

Experimental Evidence:

Comparative studies between wild-type Bcl-2 and the NWGR-deleted variant demonstrate that this domain is essential for Bcl-2's ability to form protein-protein interactions that mediate its anti-apoptotic function. The altered dimerization properties directly impact its ability to regulate mitochondrial membrane permeability and inhibit caspase activity .

What are the advantages and limitations of using Bcl-2 without the NWGR domain in experimental models?

When utilizing Bcl-2 minus NWGR domain in experimental models, researchers should consider the following advantages and limitations:

Advantages:

• Structure-Function Analysis:

  • Enables precise determination of the NWGR domain's contribution to Bcl-2 function

  • Allows identification of domain-specific interaction partners

  • Facilitates mapping of functional epitopes within Bcl-2

• Control for BH3-Mimetic Studies:

  • Provides negative control for drugs targeting the BH3-binding groove

  • Helps distinguish on-target from off-target effects of Bcl-2 inhibitors

  • Enables validation of binding specificity in drug discovery pipelines

• Novel Interaction Discovery:

  • Reveals interactions that may be masked by dominant BH3-dependent binding

  • Uncovers non-canonical functions of Bcl-2 independent of NWGR domain

  • Complements proteomic approaches that have identified proteins like galectin-7

• Technical Versatility:

  • Available as purified recombinant protein with high purity (>95%)

  • Can be detected using specific antibodies like MAB827

  • Compatible with multiple analytical techniques

Limitations:

• Structural Instability:

  • Greater tendency to form intramolecular disulfide bonds

  • Requires reducing conditions (5-10mM DTT) for proper handling

  • May adopt non-native conformations affecting experimental outcomes

• Physiological Relevance:

  • Represents an artificial variant not naturally occurring in cells

  • May not recapitulate natural regulatory mechanisms

  • Potential for artifacts when overexpressed in cellular systems

• Experimental Constraints:

  • Special storage conditions required (desiccated below -18°C)

  • Limited shelf-life after reconstitution (2-7 days at 4°C)

  • Need for carrier proteins (0.1% HSA or BSA) for long-term storage

• Interpretation Challenges:

  • Distinguishing direct effects from secondary consequences

  • Potential compensatory mechanisms in cellular models

  • Context-dependent functions across different cell types

Researchers must carefully balance these considerations when designing experiments utilizing Bcl-2 without the NWGR domain to maximize scientific insights while minimizing technical limitations.