ABSTRACT
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
Subject(s)
Apoptosis , Mitochondria/pathology , Mitochondrial Membranes/metabolism , Protein Multimerization , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , Animals , Biological Transport , Cell Membrane Permeability , Cytosol/metabolism , Humans , Mice , Mitochondria/metabolism , Models, Molecular , Protein Conformation , Proto-Oncogene Proteins c-fosABSTRACT
BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
Subject(s)
Apoptosis , bcl-2-Associated X Protein , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/genetics , Humans , Apoptosis/drug effects , Cysteine/chemistry , Cysteine/metabolism , Animals , Aldehydes/chemistry , Aldehydes/pharmacology , Models, Molecular , Mitochondria/drug effects , Mitochondria/metabolismABSTRACT
MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid ß-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1Matrix) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.
Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain/metabolism , Lipid Metabolism/physiology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Palmitic Acid/metabolism , Acyl-CoA Dehydrogenase, Long-Chain/genetics , Animals , Cell Line , Mice , Mice, Knockout , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Oxidation-Reduction , Protein Structure, SecondaryABSTRACT
Despite continuing advances in the development of novel cellular-, antibody-, and chemotherapeutic-based strategies to enhance immune reactivity, the presence of regulatory T cells (Treg cells) remains a complicating factor for their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg cell deletional strategies or small molecule drugging, we investigated the ability of hydrocarbon stapled alpha-helical (SAH) peptides to target FOXP3, the master transcription factor regulator of Treg cell development, maintenance, and suppressive function. Using the crystal structure of the FOXP3 homodimer as a guide, we developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain (SAH-FOXP3) to block this key FOXP3 protein-protein interaction (PPI) through molecular mimicry. We describe the design, synthesis, and biochemical evaluation of single- and double-stapled SAHs covering the entire coiled-coil expanse. We show that lead SAH-FOXP3s bind FOXP3, are cell permeable and nontoxic to T cells, induce dose-dependent transcript and protein level alterations of FOXP3 target genes, impede Treg cell function, and lead to Treg cell gene expression changes in vivo consistent with FOXP3 dysfunction. These results demonstrate a proof of concept for rationally designed FOXP3-directed peptide therapeutics that could be used as approaches to amplify endogenous immune responsiveness.
Subject(s)
Forkhead Transcription Factors , T-Lymphocytes, Regulatory , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Peptides/metabolism , Protein Conformation, alpha-HelicalABSTRACT
BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1-α2 loop, and α2-α3 and α5-α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism.
Subject(s)
Apoptosis , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/chemistry , bcl-2-Associated X Protein/chemistry , Amino Acid Sequence , Binding Sites/genetics , Deuterium Exchange Measurement/methods , HeLa Cells , Humans , Mass Spectrometry/methods , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Conformation , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Drug Design , Drug Resistance, Neoplasm , Humans , Molecular Conformation , Molecular Docking Simulation , Peptides/chemistry , Protein Binding , Structure-Activity Relationship , Ubiquitin/chemistry , Ubiquitin/genetics , UbiquitinationABSTRACT
BCL-2 family protein interactions regulate apoptosis, a critical process that maintains tissue homeostasis but can cause a host of human diseases when deregulated. Venetoclax is the first FDA-approved drug to reactivate apoptosis in cancer by selectively targeting an anti-apoptotic BCL-2 family member. The drug's activity relies on an 'inhibit the inhibitor' mechanism, whereby blockade of a key surface groove on BCL-2 disables its capacity to neutralize pro-apoptotic effectors, such as BAX, a chief executioner protein of the apoptotic pathway. A series of physiologic and pharmacologic regulatory sites that mediate the activation or inhibition of BAX have recently been identified, providing blueprints for the development of alternative apoptosis modulators to block pathologic cell survival or avert unwanted cell death by drugging BAX directly.
Subject(s)
Small Molecule Libraries/pharmacology , bcl-2-Associated X Protein/antagonists & inhibitors , Cell Death/drug effects , Humans , Models, Molecular , Molecular Structure , Small Molecule Libraries/chemistry , bcl-2-Associated X Protein/metabolismABSTRACT
Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1-binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1-dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or "staple" that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.
Subject(s)
Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplasms/metabolism , Peptides/chemistry , Animals , Binding Sites , Cell Line , Cell Survival , Circular Dichroism , Crystallography, X-Ray , Cytoplasm/metabolism , Drug Design , Humans , Hydrophobic and Hydrophilic Interactions , Inhibitory Concentration 50 , Mice , Mutation , Protein Binding , Protein Interaction Mapping , Spectrometry, FluorescenceABSTRACT
The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb(-/-);p53(-/-) tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive.
Subject(s)
Apoptosis/genetics , Gene Expression Regulation, Neoplastic , Mitochondria/metabolism , Retinoblastoma Protein/metabolism , Animals , Cell Line, Tumor , Cell Nucleus/metabolism , Cytochromes c/metabolism , Humans , Mice , Mice, Nude , Mice, SCID , Mitochondria/genetics , Protein Binding , Retinoblastoma Protein/genetics , Transplantation, Heterologous , Tumor Necrosis Factor-alpha/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
In this issue of Molecular Cell, Dumitru et al. (2012) report that hES cells localize a conformationally activated form of proapoptotic BAX to the trans Golgi network, a previously unanticipated launch pad for mitochondrial assault in response to DNA damage.
ABSTRACT
BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.
Subject(s)
Phenyl Ethers/pharmacology , bcl-2-Associated X Protein/chemistry , Apoptosis , Dose-Response Relationship, Drug , Drug Delivery Systems , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Phenyl Ethers/chemistry , Protein Binding , Proto-Oncogene Proteins c-bcl-2ABSTRACT
Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled-peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity.
Subject(s)
Fibroblasts/cytology , Fibroblasts/metabolism , Hydrocarbons/metabolism , Peptides/metabolism , Animals , Hydrocarbons/chemistry , Hydrocarbons/pharmacokinetics , Mice , Peptides/chemistry , Peptides/pharmacokineticsABSTRACT
Low nephron endowment at birth has been associated with an increased risk for developing hypertension and chronic kidney disease. We demonstrated in an earlier study that conditional deletion of the microRNA (miRNA)-processing enzyme Dicer from nephron progenitors results in premature depletion of the progenitors and increased expression of the proapoptotic protein Bim (also known as Bcl-2L11). In this study, we generated a compound mouse model with conditional deletion of both Dicer and Bim, to determine the biologic significance of increased Bim expression in Dicer-deficient nephron progenitors. The loss of Bim partially restored the number of nephron progenitors and improved nephron formation. The number of progenitors undergoing apoptosis was significantly reduced in kidneys with loss of a single allele, or both alleles, of Bim compared to mutant kidneys. Furthermore, 2 miRNAs expressed in nephron progenitors (miR-17 and miR-106b) regulated Bim levels in vitro and in vivo Together, these data suggest that miRNA-mediated regulation of Bim controls nephron progenitor survival during nephrogenesis, as one potential means of regulating nephron endowment.-Cerqueira, D. M., Bodnar, A. J., Phua, Y. L., Freer, R., Hemker, S. L., Walensky, L. D., Hukriede, N. A., Ho, J. Bim gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.
Subject(s)
Bcl-2-Like Protein 11/genetics , Gene Expression Regulation, Developmental/physiology , Kidney/embryology , MicroRNAs/metabolism , Animals , Apoptosis/physiology , Bcl-2-Like Protein 11/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Embryo, Nonmammalian , Gene Deletion , Gene Dosage/physiology , HEK293 Cells , Humans , Mice , Mice, Transgenic , MicroRNAs/genetics , Nephrons/cytology , Ribonuclease III/genetics , Ribonuclease III/metabolism , Stem Cells/cytology , Xenopus laevis/embryologyABSTRACT
BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.
Subject(s)
Apoptosis , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , Amino Acid Sequence , Binding Sites , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Mitochondria/metabolism , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Transport , Structure-Activity RelationshipABSTRACT
B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a BCL-2 family protein with high homology to the multidomain proapoptotic proteins BAX and BAK, yet Bok(-/-) and even Bax(-/-)Bok(-/-) and Bak(-/-)Bok(-/-) mice were reported to have no overt phenotype or apoptotic defects in response to a host of classical stress stimuli. These surprising findings were interpreted to reflect functional compensation among the BAX, BAK, and BOK proteins. However, BOK cannot compensate for the severe apoptotic defects of Bax(-/-)Bak(-/-) mice despite its widespread expression. Here, we independently developed Bok(-/-) mice and found that Bok(-/-) cells are selectively defective in their response to endoplasmic reticulum (ER) stress stimuli, consistent with the predominant subcellular localization of BOK at the ER. Whereas Bok(-/-) mouse embryonic fibroblasts exposed to thapsigargin, A23187, brefeldin A, DTT, geldanamycin, or bortezomib manifested reduced activation of the mitochondrial apoptotic pathway, the death response to other stimuli such as etoposide, staurosporine, or UV remained fully intact. Multiple organs in Bok(-/-) mice exhibited resistance to thapsigargin-induced apoptosis in vivo. Although the ER stress agents activated the unfolded protein response, both ATF4 and CHOP activation were diminished in Bok(-/-) cells and mice. Importantly, BAX and BAK were unable to compensate for the defective apoptotic response to ER stress observed in SV40-transformed and primary Bok(-/-) cells, and in vivo. These findings support a selective and distinguishing role for BOK in regulating the apoptotic response to ER stress, revealing--to our knowledge--the first bona fide apoptotic defect linked to Bok deletion.
Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum/metabolism , Oxidative Stress , Proto-Oncogene Proteins c-bcl-2/physiology , Activating Transcription Factor 4/metabolism , Animals , Annexin A5/pharmacology , Caspase 3/metabolism , Caspase 7/metabolism , Cells, Cultured , Endoplasmic Reticulum/enzymology , Enzyme Activation , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/genetics , Transcription Factor CHOP/metabolismABSTRACT
Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of â¼ 30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D(V12) activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.
Subject(s)
Drosophila Proteins/metabolism , Gene Expression Regulation, Enzymologic/physiology , MAP Kinase Signaling System/physiology , Peptides/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , SOS1 Protein/metabolism , ras Proteins/antagonists & inhibitors , ras Proteins/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Chromatography, Gel , Drosophila melanogaster , Escherichia coli , Fluorescence , Humans , MAP Kinase Signaling System/genetics , Magnetic Resonance Spectroscopy , Microfluidics , Mutation/genetics , Peptides/genetics , Peptides/metabolism , Protein Binding , Protein Structure, Secondary/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , SOS1 Protein/genetics , ras Proteins/geneticsABSTRACT
Osteosarcoma is the most common primary bone tumor, yet there have been no substantial advances in treatment or survival in three decades. We examined 59 tumor/normal pairs by whole-exome, whole-genome, and RNA-sequencing. Only the TP53 gene was mutated at significant frequency across all samples. The mean nonsilent somatic mutation rate was 1.2 mutations per megabase, and there was a median of 230 somatic rearrangements per tumor. Complex chains of rearrangements and localized hypermutation were detected in almost all cases. Given the intertumor heterogeneity, the extent of genomic instability, and the difficulty in acquiring a large sample size in a rare tumor, we used several methods to identify genomic events contributing to osteosarcoma survival. Pathway analysis, a heuristic analytic algorithm, a comparative oncology approach, and an shRNA screen converged on the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway as a central vulnerability for therapeutic exploitation in osteosarcoma. Osteosarcoma cell lines are responsive to pharmacologic and genetic inhibition of the PI3K/mTOR pathway both in vitro and in vivo.
Subject(s)
Bone Neoplasms/metabolism , Genome, Human , Osteosarcoma/metabolism , Phosphatidylinositol 3-Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Genetic Heterogeneity , Germ-Line Mutation , Humans , Osteosarcoma/genetics , Osteosarcoma/pathology , Tumor Suppressor Protein p53/geneticsABSTRACT
Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma resistant to conventional chemotherapy. Although defined by the characteristic t(11;14) translocation, MCL has not been recapitulated in transgenic mouse models of cyclin D1 overexpression alone. Indeed, several genetic aberrations have been identified in MCL that may contribute to its pathogenesis and chemoresistance. Of particular interest is the frequent biallelic deletion of the proapoptotic BCL-2 family protein BIM. BIM exerts its pro-death function via its α-helical BH3 death domain that has the dual capacity to inhibit antiapoptotic proteins such as BCL-2 and MCL-1 and directly trigger proapoptotic proteins such as the mitochondrial executioner protein BAX. To evaluate a functional role for Bim deletion in the pathogenesis of MCL, we generated cyclin D1-transgenic mice harboring Bim-deficient B cells. In response to immunization, Eµ(CycD1)CD19(CRE)Bim(fl/fl) mice manifested selective expansion of their splenic mantle zone compartment. Three distinct immune stimulation regimens induced lymphomas with histopathologic and molecular features of human MCL in a subset of mice. Thus, deletion of Bim in B cells, in the context of cyclin D1 overexpression, disrupts a critical control point in lymphoid maturation and predisposes to the development of MCL. This genetic proof of concept for MCL pathogenesis suggests an opportunity to reactivate the death pathway by pharmacologic mimicry of proapoptotic BIM.
Subject(s)
Apoptosis Regulatory Proteins/physiology , B-Lymphocytes/pathology , Cyclin D1/physiology , Lymphoma, Mantle-Cell/pathology , Membrane Proteins/physiology , Proto-Oncogene Proteins/physiology , Animals , B-Lymphocytes/metabolism , Bcl-2-Like Protein 11 , Cell Cycle , Female , Flow Cytometry , Humans , Immunoenzyme Techniques , Immunophenotyping , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesis-based solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAK-mediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3-interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.
Subject(s)
Mitochondria, Liver/chemistry , Mitochondria, Liver/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Animals , Apoptosis/physiology , Humans , Mice , Mice, Knockout , Mitochondria, Liver/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
BAX, the BCL-2-associated X protein, is a cardinal proapoptotic member of the BCL-2 family, which regulates the critical balance between cellular life and death. Because so many medical conditions can be categorized as diseases of either too many or too few cells, dissecting the biochemistry of BCL-2 family proteins and developing pharmacological strategies to target them have become high priority scientific objectives. Here, we focus on BAX, a latent, cytosolic and monomeric protein that transforms into a lethal mitochondrial oligomer in response to cellular stress. New insights into the structural location of BAX's 'on switch', and the multi-step conformational changes that ensue upon BAX activation, are providing fresh opportunities to modulate BAX for potential benefit in human diseases characterized by pathologic cell survival or unwanted cellular demise.