Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated the bcl-w gene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. The bcl-w ؊͞؊ mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.cell death | mitochondrial permeabilization | protein-membrane topology | membrane pores | cysteine-scanning mutagenesis C ommitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1-4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2-associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7-10). These novel "symmetric" homo-dimers can multimerize via association of α6 helices (8,11,12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two...

A unique combination of growth promoting factors is described that allows growth of large amounts (10(10)‐10(11)) of normal erythroid progenitors from chick bone marrow. These erythroid progenitors express the estrogen receptor (ER) as well as the receptor tyrosine kinase TGF alpha R/c‐erbB. They require both TGF alpha and estradiol for sustained self‐renewal in vitro, but terminally differentiate upon withdrawal of TGF alpha and inactivation of the ER by an antagonist (ICI 164.384). Overexpression of the human ER in erythroblasts devoid of endogenous ER revealed that the hormone‐activated ER alone arrested erythroid differentiation and repressed a large group of erythrocyte genes. When similarly overexpressed, TGF alpha R/c‐erbB inhibited the expression of a distinct, but overlapping, set of genes. The endogenous ER and TGF alpha R/c‐erbB affect erythrocyte gene expression in a similar, but less pronounced fashion. Surprisingly, suppression of ER function by antagonist efficiently inhibited erythroblast transformation by tyrosine kinase oncogenes, suggesting a role of the endogenous ER in leukemogenesis. We speculate that the oncogenes v‐erbB and v‐erbA cooperate in erythroleukemia induction by a mechanism that is employed by TGF alpha R/c‐erbB and ER to regulate normal progenitor self‐renewal in response to external signals.

To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.

Graphical Abstract Highlights d BH3 binding to BAX groove essential for late but not early steps in BAX activation d Mutations identified in BAX helices 1 and 6 near a proposed second BH3 binding site d They reduced BH3 binding to this site and stabilized BAX in inactive conformations d They revealed allosteric changes controlling BAX mitochondrial membrane association SUMMARYTo elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices a1 and a6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, a1 or a6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The a1 and a6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX a1-a2 loop allosterically sequestered its a9 membrane anchor in the groove. The crystal structure of an a6 mutant revealed additional allosteric effects. The results suggest that the a1 and a6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.(E) Upon a death stimulus, unlike the active conformation assumed by WT BAX and K21E, the groove mutants translocate to the MOM but retain an inactive conformation. Bax/Bak DKO MEFs expressing the BAX variants were pre-incubated with caspase inhibitor Q-VD.oph (25 mM) for 1 h, then treated with 5 mM etoposide. After 16 h, the cells were permeabilized with 0.025% digitonin and treated with PK (20 min on ice) before fractionation, carbonate extraction, and immunoblotting for BAX. (F) Unlike WT and K21E BAX, the groove mutants do not expose the N-terminal 6A7 epitope upon a death stimulus. DKO MEFs expressing the human BAX variants, treated as in (E), were solubilized in 1% (3-((3-cholamidopropyl) dimethylammonio)-1 (CHAPS), and activated BAX was immunoprecipitated with antibody 6A7. All immunoblots are representative of at least two independent experiments.

To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.

The conformational changes converting BAX from an inert cytosolic monomer into the homo-oligomers that permeabilize the mitochondrial outer membrane (MOM) are crucial steps toward apoptosis. Here, we have explored the potential role of the BAX α1−α2 loop in this process by three mutagenic approaches: replacing loop segments with cognate loop regions from closely related proteins, alanine scanning and analysis of BAX α1−α2 loop missense mutations observed in tumours. Responsiveness to a death signal, such as tBID, was reduced by mutations in the N-terminal but not C-terminal half of the loop. N-terminal loop variants, which were enriched in tumours, impaired MOM integration by allosterically reducing exposure of the BAX α9 transmembrane anchor. Most C-terminal loop variants reduced BAX stability, leading to increased BAX apoptotic function in some variants. Thus, our systematic mutagenesis suggests that the two halves of the α1-α2 loop have distinct functions. We show that the N-terminal half of the loop (its first nine residues) comprises an important allosteric regulator of BAX activation by setting the proportion of MOM-integrated BAX following a death signal. The enrichment of N-terminal loop mutations in tumours indicates that they may promote tumour cell survival and underscore the loop as a target for therapeutic manipulation of BAX function.

Drugs targeting various pro-survival BCL-2 family members (‘‘BH3 mimetics’’) have efficacy in hemopoietic malignancies, but the non-targeted pro-survival family members can promote resistance. Pertinently, the sensitivity of some tumor cell lines to BH3 mimetic ABT737, which targets BCL-2, BCL-XL, and BCL-W but not MCL-1, is enhanced by 2-deoxyglucose (2DG). We found that 2DG augmented apoptosis induced by ABT737 in 3 of 8 human hemopoietic tumor cell lines, most strongly in pre-B acute lymphocytic leukemia cell line NALM-6, the focus of our mechanistic studies. Although 2DG can lower MCL-1 translation, how it does so is incompletely understood, in part because 2DG inhibits both glycolysis and protein glycosylation in the endoplasmic reticulum (ER). Its glycolysis inhibition lowered ATP and, through the AMPK/mTORC1 pathway, markedly reduced global protein synthesis, as did an ER integrated stress response. A dual reporter assay revealed that 2DG impeded not only cap-dependent translation but also elongation or cap-independent translation. MCL-1 protein fell markedly, whereas 12 other BCL-2 family members were unaffected. We ascribe the MCL-1 drop to the global fall in translation, exacerbated for mRNAs with a structured 5′ untranslated region (5′UTR) containing potential regulatory motifs like those in MCL-1 mRNA and the short half-life of MCL-1 protein. Pertinently, 2DG downregulated two other short-lived oncoproteins, MYC and MDM2. Thus, our results support MCL-1 as a critical 2DG target, but also reveal multiple effects on global translation that may well also affect its promotion of apoptosis.