Brian Leber

Membrane Binding by tBid Initiates an Ordered Series of Events Culminating in Membrane Permeabilization by Bax

In normal circumstances, the Bcl-2 family dutifully governs when cells die. However, the rules of engagement between the pro- and antiapoptotic family members are still contested, and how Bax is transformed from a cytosolic monomer to an outer mitochondrial membrane-permeabilizing oligomer is unclear. With fluorescence techniques and an in vitro system, the combination of tBid and Bax produced dramatic membrane permeabilization. The membrane is not a passive partner in this process beause membranes are required for the protein-protein interactions to occur. Simultaneous measurements of these interactions revealed an ordered series of steps required for outer membrane permeabilization: (1) tBid rapidly binds to membranes, where (2) tBid interacts with Bax, causing (3) Bax insertion into membranes and (4) oligomerization, culminating in (5) membrane permeabilization. Bcl-XL prevents membrane-bound tBid from binding Bax. Bad releases tBid from Bcl-XL, restoring both tBid binding to Bax and membrane permeabilization.

Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c‐myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug‐treated c‐myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine‐specific modifying agent revealed that Bax inserted both the ‘pore domain’ (helices α5–α6), and the tail‐anchor (helix α9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc‐expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore‐forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes.

Permeabilization of the outer mitochondrial membrane is the point of no return in most programmed cell deaths. This critical step is mainly regulated by the various protein-protein and protein-membrane interactions of the Bcl-2 family proteins. The two main models for regulation of mitochondrial outer membrane permeabilization, direct activation and displacement do not account for all of the experimental data and both largely neglect the importance of the membrane. We propose the embedding together model to emphasize the critical importance of Bcl-2 family protein interactions with and within membranes. The embedding together model proposes that both pro- and anti-apoptotic Bcl-2 family proteins engage in similar dynamic interactions that are governed by membrane dependent conformational changes and culminate in either aborted or productive membrane permeabilization depending on the final oligomeric state of pro-apoptotic Bax and/or Bak.

Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types.

Human Bcl‐2 is located in multiple intracellular membranes when expressed in MDCK and Rat‐1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl‐2 carboxy‐terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation‐induced apoptosis by both wild‐type Bcl‐2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat‐1/myc cells, the Bcl‐2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild‐type, demonstrating that Bax binding is not sufficient to prevent apoptosis.

Identification of Drugs Including a Dopamine Receptor Antagonist that Selectively Target Cancer Stem Cells

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

Bcl-XL inhibits membrane permeabilization by competing with Bax.

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL–tBid and Bcl-XL–Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.

Mechanisms of Action of Bcl-2 Family Proteins

The Bcl-2 family of proteins controls a critical step in commitment to apoptosis by regulating permeabilization of the mitochondrial outer membrane (MOM). The family is divided into three classes: multiregion proapoptotic proteins that directly permeabilize the MOM; BH3 proteins that directly or indirectly activate the pore-forming class members; and the antiapoptotic proteins that inhibit this process at several steps. Different experimental approaches have led to several models, each proposed to explain the interactions between Bcl-2 family proteins. The discovery that many of these interactions occur at or in membranes as well as in the cytoplasm, and are governed by the concentrations and relative binding affinities of the proteins, provides a new basis for rationalizing these models. Furthermore, these dynamic interactions cause conformational changes in the Bcl-2 proteins that modulate their apoptotic function, providing additional potential modes of regulation.

Bcl‐2 changes conformation to inhibit Bax oligomerization

Bcl‐2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl‐2, but not the inactive point mutants Bcl‐2‐G145A and Bcl‐2‐V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl‐2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of α5–α6 helices (Bcl‐2‐S105C/E152C) was only active in a reducing environment. Thus, Bcl‐2 must change the conformation to inhibit tBid‐induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl‐2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl‐2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl‐2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane‐inserted Bax monomers and prevent productive oligomerization of Bax.

BH3-only proteins: Orchestrators of apoptosis☆

The BH3-only proteins of Bcl-2 family are essential initiators of apoptosis that propagate extrinsic and intrinsic cell death signals. The interaction of BH3-only proteins with other Bcl-2 family members is critical for understanding the core machinery that controls commitment to apoptosis by permeabilizing the mitochondrial outer membrane. BH3-only proteins promote apoptosis by both directly activating Bax and Bak and by suppressing the anti-apoptotic proteins at the mitochondria and the endoplasmic reticulum. To prevent constitutive cell death, BH3-only proteins are regulated by a variety of mechanisms including transcription and post-translational modifications that govern specific protein-protein interactions. Furthermore, BH3-only proteins also control the initiation of autophagy, another important pathway regulating cell survival and death. Emerging evidence indicates that the interaction of BH3-only proteins with membranes regulates binding to other Bcl-2 family members, thereby specifying function. Due to the important role of BH3-only proteins in the regulation of cell death, several promising BH3-mimetic drugs that are active in pre-clinical models are currently being tested as anti-cancer agents. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin

The eukaryotic translation initiation factor eIF4E is elevated in 30% of malignancies including M4/M5 subtypes of acute myeloid leukemia (AML). The oncogenic potential of eIF4E arises from its ability to bind the 7-methyl guanosine (m(7)G) cap on mRNAs, thereby selectively enhancing eIF4E-dependent nuclear mRNA export and translation. We tested the clinical efficacy of targeting eIF4E in M4/M5 AML patients with a physical mimic of the m(7)G cap, ribavirin. Among 11 evaluable patients there were 1 complete remission (CR), 2 partial remissions (PRs), 2 blast responses (BRs), 4 stable diseases (SDs), and 2 progressive diseases (PDs). Ribavirin-induced relocalization of nuclear eIF4E to the cytoplasm and reduction of eIF4E levels were associated with clinical response. Lack of response or relapse coincided with continued or renewed nuclear localization of eIF4E. This first clinical study to target eIF4E in human malignancy demonstrates clinical activity and associated molecular responses in leukemic blasts. This trial is registered at ClinicalTrials.gov (NCT00559091).