Bcl XL Human, His

What is the biological significance of BCL-XL in human cells?

BCL-XL (BCL2L1) is an essential anti-apoptotic protein belonging to the BCL-2 family, which includes BCL-2, MCL-1, A1, and BCL-W. These proteins counteract apoptotic signals that emerge during development and under stress conditions. BCL-XL was the first identified BCL-2 homologue and represents one of two splicing variants of the BCL-X gene. It functions by binding to pro-apoptotic proteins such as BIM, BMF, BAD, BIK, HRK, PUMA, tBID, and also to BAX and BAK. By shuttling BAX from mitochondria to cytosol, BCL-XL reduces BAX levels at mitochondria and decreases cellular apoptotic susceptibility .

How does recombinant His-tagged BCL-XL differ from native BCL-XL?

Recombinant His-tagged BCL-XL typically refers to E. coli-derived human BCL-XL protein (often Ser2-Arg212) with a C-terminal 6-His tag added for purification purposes. This tag allows for efficient isolation using metal affinity chromatography without significantly affecting its biological function. The recombinant protein may also lack the C-terminal transmembrane domain (minus C-terminus) to improve solubility. While functionally similar to native BCL-XL in binding assays, researchers should verify that the His-tag doesn't interfere with specific experimental applications, particularly those involving membrane interactions or structural studies .

What are optimal storage and handling conditions for His-tagged BCL-XL protein?

For optimal stability and activity, His-tagged BCL-XL protein should be stored in a manual defrost freezer, avoiding repeated freeze-thaw cycles. When shipped, the protein is typically supplied as a 0.2 μm filtered solution in HEPES and KCl and transported with dry ice or equivalent cooling. Upon receipt, it should be immediately stored at the recommended temperature. For experimental use, carrier-free versions (without BSA) are recommended for applications where BSA might interfere, while versions with carrier protein provide enhanced stability and longer shelf-life for general applications like cell culture or as ELISA standards .

What methodology should be used to assess BCL-XL inhibition in cytochrome c release assays?

To conduct cytochrome c release assays for BCL-XL inhibition studies, researchers should implement the following methodology:

• Prepare or obtain crude/enriched mouse liver mitochondria following established protocols

• Formulate appropriate buffers:

  • Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid-free BSA

  • Mitochondria Buffer: 125 mM KCl, 0.5 mM MgCl2, 3.0 mM Succinic acid, 3.0 mM Glutamic acid, 10 mM HEPES-KOH (pH 7.4), 1 mg/mL BSA, with protease inhibitors (25 μg/mL Leupeptin, 25 μg/mL Pepstatin, 3 μg/mL Aprotinin, 100 μM PMSF, and 10 μM caspase inhibitor)

• Use recombinant Human BCL-XL (minus C-Terminus) protein with optimization at concentrations <250 nM

• Include recombinant Human BID Caspase-8-cleaved (54 nM) as a positive control

• Measure cytochrome c release through appropriate detection methods such as Western blotting or ELISA

Laboratories should determine optimal dilutions for their specific experimental conditions .

How can researchers effectively design knockdown experiments to study BCL-XL function in human hematopoietic cells?

For effective BCL-XL knockdown studies in human hematopoietic cells, researchers should:

• Use lentiviral vectors expressing shRNAs targeting multiple regions of BCL-XL mRNA to ensure efficient knockdown

• Include appropriate controls:

  • Non-targeting shRNA control

  • Rescue experiments with BCL-XL overexpression constructs resistant to the shRNA

  • BCL-2 overexpression controls (as BCL-2 can compensate for BCL-XL deficiency)

• Assess knockdown efficiency through both mRNA (qRT-PCR) and protein (Western blot) analyses

• Evaluate effects at multiple stages of hematopoiesis, from early stem cells to mature blood cells

• Compare genetic knockdown effects with chemical inhibition using BH3-mimetics to validate findings

• For functional assessments, include colony formation assays, apoptosis measurements, and in vivo xenotransplantation models

This approach has revealed that BCL-XL inhibition affects human hematopoietic cells more profoundly than anticipated from murine studies, with significant losses observed from early erythropoiesis stages and reduced megakaryocyte populations .

What considerations are important when designing PROTAC molecules targeting BCL-XL?

When designing Proteolysis-Targeting Chimeras (PROTACs) directed at BCL-XL, researchers should consider these critical factors:

• Lysine accessibility: Computational modeling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase complex with BCL-XL reveals that only lysines in specific band regions can be effectively ubiquitinated, directly affecting degrader potency

• E3 ligase selection: VHL-recruiting PROTACs have successfully generated potent BCL-XL/BCL-2 dual degraders with improved antitumor activity against dependent leukemia cells

• Warhead optimization: ABT263-derived binding moieties provide effective starting points for recognition of the BH3 binding groove

• Linker design: The length, composition, and attachment point critically influence the positioning of BCL-XL relative to the E3 ligase complex, determining which lysines become accessible for ubiquitination

• Selectivity profile: Determine whether selective BCL-XL degradation or dual BCL-XL/BCL-2 targeting provides optimal therapeutic outcomes for specific indications

This structure-guided approach represents a conceptual framework for developing PROTACs with improved selectivity and potency .

How does BCL-XL dependency differ across stages of human erythropoiesis?

BCL-XL dependency in human erythropoiesis is far more extensive than previously understood from mouse models. While murine studies indicated BCL-XL importance primarily in late erythroid stages, human research reveals:

• BCL-XL is critical from the earliest stages of erythropoiesis, with efficient genetic or chemical inhibition causing significant loss of early human erythroid progenitors

• This dependency continues throughout erythroid differentiation, making the entire red blood cell production lineage vulnerable to BCL-XL inhibition

• The magnitude of BCL-XL dependency in human erythroid cells exceeds what would be predicted from murine data or early clinical trials with BCL-XL inhibitors

• BCL-XL deficiency in human erythroid precursors is not rescued by loss of its antagonist BIM, suggesting involvement of other pro-apoptotic factors

• BCL-2 overexpression can fully compensate for BCL-XL deficiency in erythroid cells, indicating potential functional redundancy between these proteins

These findings have significant implications for developing BCL-XL inhibitors as therapeutics, predicting more severe anemia than observed in early clinical trials with pan-BCL-2 family inhibitors .

What is the impact of BCL-XL inhibition on human hematopoietic stem cells and progenitors?

BCL-XL inhibition profoundly affects human hematopoietic stem cells (HSCs) and multipotent progenitors in ways not predicted by mouse studies:

• BCL-XL deficient human HSCs and progenitors show significant numerical reduction

• These cells demonstrate severely impaired capacity to engraft during xenotransplantation experiments in mice

• This contrasts with murine studies where Bcl-x deletion in adult hematopoietic cells did not noticeably affect the HSPC compartment

• BCL-XL deficiency in human HSCs can be fully compensated by BCL-2 overexpression

• Loss of the pro-apoptotic protein BIM does not rescue human HSCs from BCL-XL deficiency

These findings suggest that specific BCL-XL inhibitors may cause more severe hematological toxicities than previously anticipated, potentially affecting not only mature blood cells but also the stem cell compartment .

How do megakaryocytes and platelets depend on BCL-XL for survival?

Megakaryocytes and platelets show significant BCL-XL dependency through several mechanisms:

• Clinical studies with Navitoclax (ABT-263), a combined BCL-2/BCL-XL/BCL-W inhibitor, demonstrate severe thrombocytopenia caused by direct platelet destruction

• This thrombocytopenia is partially counteracted by increased megakaryopoiesis, suggesting a compensatory response

• Genetic BCL-XL inhibition studies confirm reduction in megakaryocyte populations

• Platelets are particularly vulnerable to BCL-XL inhibition due to their inability to synthesize new proteins and limited alternative survival mechanisms

• The platelet dependency on BCL-XL represents a primary dose-limiting toxicity for BCL-XL-targeting therapeutics

These findings explain the clinical observation of thrombocytopenia with BCL-XL inhibitors and highlight the challenge of developing such agents with acceptable therapeutic windows .

How can researchers reconcile differences between mouse and human BCL-XL dependency patterns?

The significant discrepancies between mouse and human BCL-XL dependency patterns require careful analysis:

• Experimental approaches:

  • Conduct direct comparative studies using identical methodologies

  • Perform comprehensive expression profiling of all BCL-2 family members across species and cell types

  • Utilize humanized mouse models to better recapitulate human hematopoiesis

• Mechanistic considerations:

  • Examine species-specific differences in expression patterns of other anti-apoptotic proteins that might compensate for BCL-XL in mice but not humans

  • Investigate variations in mitochondrial priming states between species

  • Analyze differences in upstream regulatory pathways controlling BCL-XL function

• Interpretation guidelines:

  • Exercise caution when extrapolating murine findings to human therapeutic applications

  • Prioritize data from human primary cells and humanized models for clinical development decisions

  • Consider species differences when establishing dosing strategies and toxicity monitoring

These reconciliation approaches are essential for accurate prediction of therapeutic outcomes and side effect profiles when targeting BCL-XL in humans .

What molecular mechanisms explain why BCL-XL deficiency affects human HSCs differently than mouse HSCs?

The differential impact of BCL-XL deficiency on human versus mouse HSCs involves several potential molecular mechanisms:

• Expression profile differences:

  • Human HSCs may have different basal levels of pro-apoptotic BCL-2 family members

  • The compensatory capacity of other anti-apoptotic proteins likely differs between species

• Cellular stress handling:

  • Human and mouse HSCs may experience different levels of metabolic or replicative stress

  • Species-specific differences in DNA damage response pathways may contribute

• Mitochondrial dynamics:

  • Human HSCs may have different mitochondrial priming states (proximity to apoptotic threshold)

  • Species-specific differences in mitochondrial structure or function could influence BCL-XL dependency

• Microenvironmental factors:

  • Differences in niche interactions between human and mouse HSCs may affect their reliance on anti-apoptotic proteins

  • Cytokine responsiveness varies between species, potentially altering survival requirements

The finding that BCL-2 overexpression can rescue BCL-XL deficiency, while BIM deletion cannot, suggests complex mechanisms involving multiple proteins beyond a simple BCL-XL/BIM axis .

How does lysine accessibility influence PROTAC-mediated BCL-XL degradation?

Lysine accessibility plays a critical role in determining the efficacy of PROTAC-mediated BCL-XL degradation:

Which hematological malignancies represent promising targets for BCL-XL inhibition?

Based on the established role of BCL-XL in human hematopoiesis, several hematological malignancies emerge as potential candidates for BCL-XL-targeting therapeutics:

• Polycythemia vera: This myeloproliferative neoplasm characterized by excessive red blood cell production may be sensitive to BCL-XL inhibition given the critical dependency of the erythroid lineage on BCL-XL

• Acute erythroid leukemia: Malignant transformation of erythroid precursors likely preserves or enhances BCL-XL dependency, potentially making these rare leukemias particularly vulnerable

• Essential thrombocytosis: Although platelets are highly BCL-XL dependent, malignant megakaryocytes driving excessive platelet production may represent therapeutic targets if selective delivery can be achieved

• Acute megakaryocytic leukemia: The megakaryocytic origin of these leukemias suggests potential sensitivity to BCL-XL inhibition

• BCL-XL-dependent lymphoid malignancies: Certain lymphomas and leukemias may exhibit BCL-XL dependency despite originating from lineages where BCL-2 typically predominates

Therapeutic success will require balancing efficacy against the expected hematological toxicities, particularly thrombocytopenia and anemia .

How can researchers assess and predict potential hematological toxicities of novel BCL-XL inhibitors?

To effectively assess and predict hematological toxicities of novel BCL-XL inhibitors, researchers should implement a comprehensive evaluation strategy:

• In vitro assessment:

  • Test effects on primary human HSCs, erythroid precursors, and megakaryocytes at multiple differentiation stages

  • Compare with established inhibitors like Navitoclax to benchmark toxicity profiles

  • Evaluate concentration-dependent effects to establish therapeutic windows

• Ex vivo systems:

  • Utilize whole blood assays to assess platelet survival

  • Implement colony formation assays with primary human bone marrow

  • Develop organoid or 3D culture systems that better recapitulate bone marrow niches

• In vivo models:

  • Employ humanized mouse models with human hematopoietic system reconstitution

  • Monitor multiple hematological parameters including stem cell function

  • Assess long-term effects on hematopoietic recovery

• Biomarker development:

  • Identify early indicators of hematological toxicity

  • Establish correlation between biomarker changes and functional hematological outcomes

These approaches will provide more accurate predictions of human toxicities than conventional mouse models alone, given the demonstrated species differences in BCL-XL dependency patterns .

What strategies might improve the therapeutic window of BCL-XL-targeting compounds?

Several innovative approaches may enhance the therapeutic window of BCL-XL-targeting compounds:

• Targeted delivery systems:

  • Antibody-drug conjugates targeting malignancy-specific antigens

  • Nanoparticle formulations with preferential tumor accumulation

  • Tumor microenvironment-activated prodrugs

• Advanced drug design:

  • Dual BCL-XL/BCL-2 inhibitors optimized for specific malignancies

  • Development of PROTACs that achieve catalytic degradation, potentially requiring lower drug exposure

  • Tissue-selective E3 ligase recruiting moieties to direct degradation preferentially in malignant cells

• Combination strategies:

  • Pairing with agents that selectively sensitize malignant cells

  • Identifying synergistic combinations allowing lower BCL-XL inhibitor doses

  • Temporary cytoprotection of normal hematopoietic cells during treatment

• Modified administration approaches:

  • Optimized dosing schedules allowing hematological recovery between treatments

  • Pulsatile high-concentration exposure rather than continuous dosing

  • Pre-emptive growth factor support to counteract specific toxicities

These strategies aim to maintain therapeutic efficacy while minimizing the impact on normal hematopoietic cells that depend on BCL-XL for survival .