BCL2L2 Human, His

What is the basic structure of recombinant BCL2L2 Human with His Tag?

BCL2L2 Human Recombinant produced in E. Coli is a single, non-glycosylated polypeptide chain containing 192 amino acids (1-172 a.a.) with a molecular weight of 20.9kDa. The protein is fused to a 20 amino acid His-Tag at the N-terminus and is purified using proprietary chromatographic techniques. The complete amino acid sequence includes: MGSSHHHHHH SSGLVPRGSH MATPASAPDT RALVADFVGY KLRQKGYVCG AGPGEGPAAD PLHQAMRAAG DEFETRFRRT FSDLAAQLHV TPGSAQQRFT QVSDELFQGG PNWGRLVAFF VFGAALCAES VNKEMEPLVG QVQEWMVAYL ETRLADWIHS SGGWAEFTAL YGDGALEEAR RLREGNWASV RT .

What are the primary molecular mechanisms through which BCL2L2 regulates apoptosis?

BCL2L2 functions primarily by forming hetero- or homodimers with other members of the BCL-2 protein family to regulate the intrinsic apoptosis pathway. Specifically, BCL2L2 promotes cell survival by suppressing the death-promoting activity of pro-apoptotic proteins such as BAX. In megakaryocytes, BCL2L2 restrains apoptosis, which enables proper development and proplatelet formation. Mechanistically, BCL2L2 maintains mitochondrial membrane integrity, preventing cytochrome c release and subsequent caspase activation that would otherwise trigger cell death .

How does BCL2L2 differ functionally from other BCL-2 family members?

While BCL2L2 shares the anti-apoptotic functions of other BCL-2 family members, it demonstrates tissue-specific roles that distinguish it from related proteins. Unlike BCL-2 and BCL-XL, BCL2L2 plays a particularly important role in adult spermatogenesis and in the survival of specific neuronal populations dependent on NGF and BDNF. In megakaryocyte biology, BCL2L2 has been shown to not only prevent apoptosis but also promote proplatelet formation, suggesting a dual role in both survival and differentiation that may not be shared by all anti-apoptotic BCL-2 family members .

What are the optimal storage conditions for maintaining BCL2L2 protein stability in research applications?

For maintaining BCL2L2 protein stability, researchers should store the protein at 4°C if the entire vial will be used within 2-4 weeks. For longer periods, store frozen at -20°C. To enhance stability during long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA). The formulation containing 20mM Tris-HCl (pH-8), 100mM NaCl, and 10% glycerol provides optimal stability. Multiple freeze-thaw cycles should be strictly avoided as they can compromise protein integrity and activity. Purity checks should be performed using SDS-PAGE, with expected purity greater than 95.0% .

What lentiviral vector systems are most effective for BCL2L2 overexpression studies in human cell lines?

For effective BCL2L2 overexpression studies, mammalian gene expression lentiviral vectors have demonstrated high efficiency. Research with cord blood-derived megakaryocytes utilized lentiviral vectors containing BCL2L2, resulting in significant increases in both mRNA and protein expression by day 13 of culture. When designing lentiviral constructs, researchers should include appropriate promoters (such as CMV or EF1α) for sustained expression in their target cell type. For quantifying overexpression efficacy, both qRT-PCR for mRNA levels and western blotting for protein expression should be implemented, as demonstrated in studies where both measurements confirmed successful transduction .

What flow cytometry protocols best quantify the anti-apoptotic effects of BCL2L2 in hematopoietic cells?

To quantify the anti-apoptotic effects of BCL2L2 in hematopoietic cells, flow cytometry using Annexin V and CD41a co-staining has proven effective. In megakaryocyte research, this approach allows for specific analysis of apoptosis in the megakaryocyte lineage. The protocol should include:

• Harvesting cells from culture at appropriate time points (day 13-14 for megakaryocytes)

• Staining with CD41a antibody (megakaryocyte marker)

• Counter-staining with Annexin V to identify apoptotic cells

• Including a viability dye (e.g., 7-AAD) to distinguish early apoptotic from late apoptotic/necrotic cells

• Analyzing the percentage of Annexin V+ CD41a+ cells to measure apoptosis rate

This approach has successfully demonstrated that BCL2L2 overexpression significantly reduces the percentage of Annexin V+ CD41a+ megakaryocytes, confirming its anti-apoptotic function .

How does BCL2L2 regulate megakaryocyte development and proplatelet formation?

BCL2L2 plays a dual role in megakaryocyte biology by both restraining apoptosis and promoting proplatelet formation. Experimental evidence shows that BCL2L2 overexpression decreases megakaryocyte apoptosis, measured by reduced Annexin V positivity in CD41a+ cells. This anti-apoptotic effect results in increased numbers of CD41a+ large and low granularity (LLG) megakaryocytes by approximately 19% (from 1.36×10^5 to 1.61×10^5; P=0.049). More significantly, BCL2L2 overexpression increases proplatelet formation by 58%. The mechanism appears to involve extended megakaryocyte survival during the critical maturation phase, allowing more cells to reach the proplatelet-forming stage. Additionally, BCL2L2 may influence cytoskeletal reorganization necessary for proplatelet extension, though this requires further mechanistic investigation .

What is the correlation between BCL2L2 expression and platelet counts in healthy donors?

A significant positive correlation exists between platelet BCL2L2 mRNA levels and platelet counts in healthy donors. In an association study involving 154 healthy individuals (the Platelet RNA Expression Study 1, PRAX-1), researchers identified that higher BCL2L2 expression in platelets correlated with increased platelet numbers. This finding aligns with experimental data showing that cultured megakaryocytes overexpressing BCL2L2 produced more platelet-like particles. The relationship was assessed using Pearson's correlation with 95% Confidence Interval. This correlation suggests BCL2L2 may be a physiological regulator of platelet production in humans, though causality requires further investigation through longitudinal studies .

How does inhibition of BCL2L2 affect cultured megakaryocyte yield and functionality?

Inhibition of BCL2L2, along with other Bcl-2 family members, significantly reduces megakaryocyte yields in culture systems. When treated with the general Bcl-2 family inhibitor ABT-263, cultures show a marked reduction in CD41a+ LLG megakaryocyte numbers. This suggests that BCL2L2 activity is necessary for optimal megakaryocyte survival and development in vitro. Furthermore, functional studies indicate that BCL2L2 influences not only megakaryocyte survival but also platelet functionality, as BCL2L2 overexpression induces small but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. These findings position BCL2L2 as a potential target for enhancing megakaryocyte yields in vitro for both research purposes and potential clinical applications in platelet production .

What evidence supports BCL2L2 as a target for the 14q11.2 amplification in non-small cell lung cancer?

Multiple lines of evidence support BCL2L2 as a target gene for the 14q11.2 amplification in non-small cell lung cancer (NSCLC). First, fluorescence in situ hybridization (FISH) analyses identified significant amplification with double minute chromosome patterns at the 14q11.2 region in NSCLC cell lines, specifically HUT29. The amplified region spans approximately 1 Mb between RP11-137H15 and RP11-76P17, containing at least 28 genes. Among these candidates, BCL2L2 was identified as the most likely target based on: (1) significant overexpression in the HUT29 cell line with amplification, (2) relatively frequent overexpression in additional NSCLC cell lines compared to normal lung epithelial cells, and (3) its known function as a pro-survival regulator that inhibits cell death. Further supporting its role, BCL2L2 is expressed frequently in human tumor cells and primary adenocarcinomas, suggesting its potential significance in cancer development and progression .

How does BCL2L2 overexpression correlate with clinical features and prognosis in lung adenocarcinoma?

Immunohistochemical analysis of 61 primary cases of lung adenocarcinoma demonstrated that BCL2L2 overexpression is significantly associated with tumor stage and differentiation status. The analysis graded BCL2L2 expression as positive when tumor-cell cytoplasm showed higher immunopositivity compared with normal alveolar type II cells (considered to be the source of lung adenocarcinomas) or negative when tumor-cell cytoplasm showed immunonegativity compared to these normal cells. Results indicated that 40 out of 61 cases (65.6%) showed BCL2L2 protein overexpression. While the data showed varying associations with clinical parameters, BCL2L2 overexpression tended to be associated with a poorer prognosis. This suggests BCL2L2 may serve as a prognostic biomarker in lung adenocarcinoma and potentially a therapeutic target for intervention strategies .

What are the key considerations when designing siRNA-mediated knockdown experiments for BCL2L2?

When designing siRNA-mediated knockdown experiments for BCL2L2, researchers should consider several critical factors:

• Target sequence selection: Design at least 3-4 different siRNA sequences targeting different regions of BCL2L2 mRNA to ensure successful knockdown and control for off-target effects

• Appropriate controls: Include non-targeting control siRNAs with similar GC content and a positive control siRNA targeting a housekeeping gene

• Transfection optimization: Determine optimal transfection conditions for your specific cell type, as transfection efficiency varies significantly between cell lines

• Knockdown validation: Verify knockdown efficiency at both mRNA level (qRT-PCR) and protein level (Western blot), as BCL2L2 protein may have a longer half-life than its mRNA

• Timing considerations: Assess phenotypes at multiple time points after transfection, as the kinetics of BCL2L2 depletion and subsequent cellular effects may vary

• Functional readouts: Include appropriate apoptosis assays (Annexin V/PI staining, caspase activation) as well as cell-type specific functional assays

As demonstrated in cancer research studies, successful BCL2L2 knockdown can reveal its role in cell growth and survival, providing valuable insights into its potential as a therapeutic target .

How can researchers distinguish between the effects of BCL2L2 and other BCL-2 family members in experimental systems?

Distinguishing between the effects of BCL2L2 and other BCL-2 family members requires a multi-faceted approach:

• Selective inhibition: While pan-BCL-2 family inhibitors like ABT-263 affect all members, researchers should compare these results with selective targeting approaches:

  • Specific siRNA or shRNA against BCL2L2

  • CRISPR/Cas9-mediated knockout of BCL2L2

  • Selective small molecule inhibitors (where available)

• Rescue experiments: After knockdown or inhibition, perform rescue experiments with:

  • BCL2L2-specific overexpression

  • Mutated versions resistant to the inhibition strategy

  • Other BCL-2 family members to test functional redundancy

• Expression profiling: Quantify the relative expression levels of all BCL-2 family members in your experimental system to determine which members might compensate for BCL2L2 loss

• Protein interaction studies: Use co-immunoprecipitation or proximity ligation assays to identify the specific binding partners of BCL2L2 compared to other family members

• Domain-specific mutations: Introduce mutations in BCL2L2-specific domains to identify unique functional regions not shared with other family members

This comprehensive approach helps delineate the unique contributions of BCL2L2 to cellular phenotypes versus those attributable to other BCL-2 family proteins .

What are the methodological challenges in studying BCL2L2 in primary human megakaryocyte cultures?

Studying BCL2L2 in primary human megakaryocyte cultures presents several methodological challenges:

• Cell source heterogeneity: Cord blood-derived megakaryocyte cultures produce heterogeneous populations with varying maturation stages, complicating analysis. Researchers should employ flow cytometry to distinguish distinct populations (e.g., large low-granularity vs. small high-granularity megakaryocytes).

• Culture optimization: Achieving sufficient megakaryocyte yield and maturation requires optimized cytokine cocktails and culture conditions. The culture system must support both proliferation and terminal differentiation phases.

• Apoptosis as a confounding factor: Since apoptosis naturally occurs during megakaryocyte development, distinguishing pathological from physiological apoptosis requires careful experimental design with appropriate controls.

• Transfection/transduction efficiency: Primary megakaryocytes are difficult to transfect directly. Researchers typically must transduce hematopoietic progenitors before differentiation, requiring optimization of viral vectors and transduction protocols.

• Functional assessment complexity: Proplatelet formation, the ultimate functional readout, is technically challenging to quantify and standardize across experiments.

• Limited material: Primary cells have finite proliferation capacity, necessitating careful experimental planning to maximize data acquisition from limited material.

These challenges necessitate robust protocols and careful interpretation of results when studying BCL2L2's role in megakaryopoiesis .

How might BCL2L2 be targeted therapeutically in disorders of platelet production?

BCL2L2 represents a promising therapeutic target for disorders of platelet production based on its demonstrated role in megakaryocyte survival and proplatelet formation. For thrombocytopenia (low platelet count conditions), strategies to enhance BCL2L2 function could include:

• Gene therapy approaches: Lentiviral vectors expressing BCL2L2 could be used to transduce hematopoietic stem cells before transplantation in severe refractory thrombocytopenia cases

• Small molecule activators: Development of compounds that enhance BCL2L2 activity or expression to promote megakaryocyte survival

• miRNA inhibitors: Anti-miRNAs targeting BCL2L2-suppressing microRNAs could increase BCL2L2 expression

• Ex vivo platelet generation: BCL2L2 overexpression systems could enhance the yield of platelets generated from stem cells for transfusion purposes

Conversely, for thrombocytosis or essential thrombocythemia (elevated platelet conditions), BCL2L2 inhibition strategies might include:

• Selective BCL2L2 inhibitors: Development of compounds specifically targeting BCL2L2 without affecting other BCL-2 family members

• siRNA/shRNA therapeutic delivery: Targeted delivery systems to reduce BCL2L2 expression in megakaryocytes

• Peptide inhibitors: Design of peptides that disrupt BCL2L2's anti-apoptotic interactions

Any therapeutic application would require careful consideration of tissue specificity to avoid unwanted effects in other BCL2L2-dependent tissues like neurons and testicular cells .

What role might BCL2L2 play in resistance to cancer therapies targeting other BCL-2 family members?

BCL2L2 may contribute significantly to resistance against cancer therapies targeting other BCL-2 family members through several mechanisms:

• Compensatory upregulation: When BCL-2 or BCL-XL are inhibited by targeted therapies such as venetoclax (ABT-199), cancer cells may adaptively upregulate BCL2L2 expression as a compensatory survival mechanism

• Differential binding profile: BCL2L2 has a distinct binding profile with pro-apoptotic BCL-2 family members compared to BCL-2 or BCL-XL, potentially allowing it to neutralize pro-apoptotic proteins not effectively targeted by existing therapies

• Amplification events: The 14q11.2 amplification observed in some cancers suggests genetic mechanisms for BCL2L2 overexpression that could drive resistance to therapies targeting other family members

• Tissue-specific effects: BCL2L2's unique tissue-specific roles might make it particularly important in resistance development in certain cancer types, especially those derived from tissues where BCL2L2 plays physiologically important roles

Understanding these resistance mechanisms could inform the development of combination therapies that simultaneously target multiple BCL-2 family members, including BCL2L2, to prevent resistance development. Monitoring BCL2L2 expression levels before and during treatment might also help predict therapeutic response and guide treatment decisions .

What emerging technologies are advancing our understanding of BCL2L2 protein interactions and regulatory networks?

Several cutting-edge technologies are enhancing our understanding of BCL2L2's interactions and regulatory networks:

• Proximity-based proteomics: BioID or APEX2-based approaches are being used to map the complete interactome of BCL2L2 in living cells, revealing both known and novel protein interactions that mediate its anti-apoptotic function

• CRISPR screens: Genome-wide CRISPR-Cas9 screens are identifying synthetic lethal interactions with BCL2L2, revealing genes whose loss is particularly toxic in BCL2L2-dependent cells

• Single-cell technologies: Single-cell RNA-seq and proteomics are uncovering cell-to-cell variability in BCL2L2 expression and function, particularly important in heterogeneous systems like megakaryocyte cultures and tumor samples

• Structural biology advances: Cryo-EM and advanced NMR techniques are providing unprecedented structural insights into BCL2L2's interactions with other proteins, facilitating structure-based drug design

• Protein engineering approaches: Engineered variants of BCL2L2 with modified binding specificities are being used to dissect the relative importance of different protein-protein interactions

• Advanced viral vector systems: Improved lentiviral, adenoviral, and AAV vector systems are enabling more precise manipulation of BCL2L2 expression in diverse experimental settings, including in vivo models

These technologies collectively promise to provide a more comprehensive understanding of BCL2L2's role in normal physiology and disease, potentially leading to novel therapeutic strategies .

How should researchers interpret conflicting data on BCL2L2 expression in different cancer types?

When confronted with conflicting data regarding BCL2L2 expression across different cancer types, researchers should consider several factors in their interpretation:

• Tissue context specificity: BCL2L2 may play different roles depending on tissue origin. Its physiological expression in normal tissues varies, so its significance when overexpressed will likely differ between cancer types.

• Methodological differences: Variations in detection methods (IHC, qRT-PCR, RNA-seq) can yield apparently conflicting results. Antibody specificity, RNA integrity, and quantification methods should be critically evaluated.

• Sample heterogeneity: Tumor heterogeneity and the presence of stromal cells can confound expression analyses. Single-cell approaches or microdissection techniques may clarify conflicting bulk tumor data.

• Genetic background: The functional impact of BCL2L2 may depend on the constellation of other genetic alterations present in different tumors. For example, its significance may differ in p53-mutant versus p53-wild-type contexts.

• Stage-dependent effects: BCL2L2's role may evolve during cancer progression. Researchers should stratify analyses by disease stage when possible.

• Correlation versus causation: High expression alone doesn't prove functional significance. Functional studies (knockdown/overexpression) in appropriate models are essential to resolve contradictory observational data.

Researchers should attempt to reconcile conflicting data through meta-analyses, standardized methodologies, and comprehensive functional validation in multiple model systems .

What statistical approaches are most appropriate for analyzing BCL2L2 expression in relation to clinical outcomes?

When analyzing BCL2L2 expression in relation to clinical outcomes, researchers should employ these statistical approaches:

• Survival analysis:

  • Kaplan-Meier curves with log-rank tests to compare survival between BCL2L2-high and BCL2L2-low groups

  • Cox proportional hazards modeling to adjust for confounding variables (age, stage, etc.)

  • Time-dependent ROC curve analysis to evaluate BCL2L2's predictive performance at different timepoints

• Expression threshold determination:

  • ROC curve analysis to determine optimal cut-off values for categorizing BCL2L2 expression

  • Minimum p-value approach with appropriate correction for multiple testing

  • X-tile analysis for visual determination of optimal thresholds

• Correlation studies:

  • Pearson's or Spearman's correlation for continuous variables (e.g., BCL2L2 mRNA levels vs. platelet counts)

  • Appropriate tests for categorical variables (Chi-square, Fisher's exact test)

• Multivariable modeling:

  • Regression models (linear, logistic, Cox) incorporating BCL2L2 with established prognostic factors

  • Interaction terms to identify effect modification

• Validation approaches:

  • Internal validation (bootstrap, cross-validation)

  • External validation in independent cohorts

  • Sensitivity analyses with alternative cutpoints or analytical methods

These approaches should be selected based on sample size, data distribution, endpoint definition, and study objectives. Researchers should clearly report all statistical methods, justify their choices, and acknowledge limitations .

What are the most promising avenues for future BCL2L2 research in hematology?

Several promising research directions for BCL2L2 in hematology warrant further investigation:

• Ex vivo platelet production optimization: Building on findings that BCL2L2 increases megakaryocyte proplatelet formation by 58%, optimized expression systems could enhance ex vivo platelet production for transfusion medicine.

• Single-cell analysis of megakaryocyte differentiation: Single-cell transcriptomics and proteomics during megakaryopoiesis could reveal how BCL2L2 expression changes throughout differentiation and identify co-regulated genes.

• BCL2L2 in thrombopoietic disorders: Investigation of BCL2L2 expression and genetic variants in patients with inherited or acquired thrombocytopenia could identify subgroups that might benefit from BCL2L2-targeted therapies.

• Regulatory mechanisms: Identifying transcriptional, post-transcriptional, and post-translational regulators of BCL2L2 in megakaryocytes could reveal new therapeutic targets.

• Interaction with thrombopoietin signaling: Exploring cross-talk between BCL2L2 and thrombopoietin receptor (c-MPL) signaling pathways could uncover synergistic approaches for enhancing platelet production.

• BCL2L2 in hematopoietic stem cell maintenance: Investigating BCL2L2's role in hematopoietic stem cell survival and self-renewal could have implications for bone marrow transplantation and hematopoietic recovery.

These research directions could significantly advance our understanding of platelet production biology and lead to novel therapeutic approaches for various hematological disorders .

How might BCL2L2 research contribute to the development of next-generation cancer therapeutics?

BCL2L2 research has significant potential to contribute to next-generation cancer therapeutics through several avenues:

• Selective BCL2L2 inhibitors: Development of small molecules specifically targeting BCL2L2 could address cancers resistant to existing BCL-2 family inhibitors like venetoclax, particularly in tumors where BCL2L2 amplification has been identified (e.g., the 14q11.2 amplicon in lung cancer).

• Combination therapy rationales: Understanding BCL2L2's interaction network could reveal synthetic lethal combinations where BCL2L2 inhibition synergizes with other targeted therapies or conventional chemotherapeutics.

• Biomarker development: BCL2L2 expression or amplification could serve as a biomarker for patient stratification, identifying those who might benefit from specific therapeutic approaches.

• Overcoming resistance mechanisms: Elucidating how cancer cells upregulate BCL2L2 to evade therapy could lead to rational combination strategies that prevent resistance development.

• Dual-targeting approaches: Designing molecules that simultaneously inhibit multiple BCL-2 family members, including BCL2L2, could provide broader efficacy across diverse cancer types.

• BCL2L2-targeted antibody-drug conjugates: Exploiting differential expression patterns of BCL2L2 could enable the development of antibody-drug conjugates for selective targeting of cancer cells.

These approaches could expand the therapeutic arsenal against cancers that currently have limited treatment options, particularly those with intrinsic or acquired resistance to existing apoptosis-targeting therapies .

What technological advances are needed to better understand BCL2L2's role in tissue-specific apoptosis regulation?

Advancing our understanding of BCL2L2's tissue-specific roles in apoptosis regulation will require several technological innovations:

• Tissue-specific conditional knockout models: More sophisticated genetic tools allowing temporal and spatial control of BCL2L2 expression in specific cell types would help delineate its function in complex tissues without developmental confounders.

• Intravital imaging of apoptosis dynamics: Advanced microscopy techniques allowing real-time visualization of BCL2L2 activity and apoptosis in living tissues would provide insights into its temporal regulation.

• Protein interaction mapping in native contexts: Improved techniques for capturing protein-protein interactions in their native cellular environment and in specific tissues would reveal tissue-specific BCL2L2 binding partners.

• BCL2L2 conformational biosensors: Development of sensors that report on BCL2L2's activation state in living cells would help understand its regulation in different tissues.

• Spatial transcriptomics and proteomics: Technologies that preserve spatial information while analyzing gene and protein expression would reveal BCL2L2's role in tissues with complex architecture.

• Targeted protein degradation approaches: Tissue-specific proteolysis targeting chimeras (PROTACs) or similar technologies would enable acute and selective BCL2L2 depletion in specific tissues.

• Organoid models: Advanced organoid culture systems representing different tissues would provide more physiologically relevant contexts for studying BCL2L2 function than traditional cell lines.