Puma strikes Bax

The commitment to programmed cell death via apoptosis is largely made upon activation of the proapoptotic mitochondrial proteins Bax or Bak. In this issue, Gallenne et al. (Gallenne, C., F. Gautier, L. Oliver, E. Hervouet, B. Noël, J.A. Hickman, O. Geneste, P.-F. Cartron, F.M. Vallette, S. Manon, and P. Juin. 2009. J. Cell Biol. 185:279–290) provide evidence that the p53 up-regulated modulator of apoptosis (Puma) protein can directly activate Bax.The Bcl-2 family of proteins participates in the control of the cell''s commitment to programmed cell death via the mitochondrial or intrinsic apoptotic pathway. Certain proteins in this family, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl-1/A1, inhibit apoptosis, whereas others in this family promote apoptosis. Proapoptotic Bax and Bak appear to be indispensible for apoptosis (Lindsten et al., 2000; Wei et al., 2001). How does the cell determine fate in the face of competing pro- and antiapoptotic proteins? The rheostat model proposed that when there were more antiapoptotic proteins than proapoptotic proteins, the cell survived and vice versa. However, in many cases, the conversion of a living cell to one committed to death occurs without significant change in the levels of pro- and antiapoptotic proteins. The participation of a third class of proapoptotic proteins largely explained this riddle. These proteins, so-called BH3-only as they share homology only in the proapoptotic Bcl-2 homology 3 domain, appear to act as sentinels of cell damage, which convert initial perturbations into death signals, that act in the mitochondrial pathway. Now, Gallenne et al. (see p. 279 of this issue) provide mechanistic insight into how the BH3-only protein Puma promotes apoptosis. The authors find that Puma, like the BH3-only proteins Bim and Bid, directly activates Bax.A key event in the commitment to apoptosis is Bax- and Bak-mediated permeabilization of the outer mitochondrial membrane. For this to occur, Bax and Bak alter their conformation from an inactive to an active form, form homo-oligomers in the membrane, and contribute to the formation of pores, which allows the egress of proapoptotic proteins to the cytosol (Fig. 1). Although there is consensus that Bax and Bak must shift from an inactive to an active state for this to occur, there is less consensus about what specific factors cause this crucial switch (Willis et al., 2007). Bid and Bim have been shown to cause activation (conformational change and oligomerization) of Bax and Bak in cellular, mitochondrial, and liposomal systems (Wei et al., 2000; Kuwana et al., 2002; Cartron et al., 2004; Certo et al., 2006). Direct interaction between these activators and Bax has been established experimentally (Gavathiotis et al., 2008; Lovell et al., 2008). Additional studies have suggested that p53 itself may translocate to the mitochondria and activate Bax after select stimuli (Mihara et al. 2003; Chipuk et al., 2004). Even heat has been indicted as a potential activating factor (Pagliari et al., 2005). It is quite possible that many activating factors remain to be discovered.Open in a separate windowFigure 1.Control of mitochondrial permeabilization by Bcl-2 family proteins. Activated Bax or Bak are available to oligomerize either when they are directly activated by activating factors, including activator BH3-only proteins (top), or when preactivated Bax or Bak are displaced from antiapoptotic proteins by either activator or sensitizer BH3-only proteins (bottom). Gallenne et al. (2009) provide evidence that Puma is an activator rather than a sensitizer. Oligomerized Bax or Bak participate in forming a pore that allows egress of proapoptotic factors like cytochrome c. Cytochrome c promotes formation of the apoptosome complex, which causes activation of effector caspases. These proteases cleave many key cellular proteins to bring about the apoptotic phenotype. Figure adapted with permission from the Journal of Cell Science (Brunelle, J.K., and A. Letai. 2009. J. Cell Sci. 122:437–441).Antiapoptotic proteins inhibit apoptosis by binding proapoptotic factors. In many cases, the proapoptotic factors are activator BH3-only proteins like Bid and Bim. However, in some cases, the proapoptotic factors may also include activated monomeric Bax and Bak, which are intercepted before they can oligomerize and form pores. Cells have been described in which antiapoptotic proteins are loaded with abundant prodeath proteins as being “primed for death.” Such cells are particularly sensitive to treatment with chemotherapy and antagonists of antiapoptotic proteins like ABT-737 (Certo et al., 2006; Deng et al., 2007). In most cells, the vast majority of Bax and Bak are in the inactive form, and activated Bax and Bak can be difficult to detect in the absence of toxic perturbation. Nonetheless, BH3-only molecules, which lack the ability to directly activate Bax or Bak, can cause apoptosis by competing for binding to antiapoptotic proteins (Fig. 1). If this competition frees sufficient activator proteins (or activated Bax and Bak), oligomerization of Bax and Bak ensues, committing the cell to death. Based on performance in assays on mitochondria and artificial liposomes spiked with Bax, the BH3-only family has thus been segregated into two subfamilies: the sensitizers and the activators.Where does Puma fit in? Puma was initially identified as a p53-regulated gene that was induced after DNA damage (Nakano and Vousden, 2001). It has subsequently been found that Puma is responsible for much of the proapoptotic effect of p53 induction but that Puma can also cause apoptosis in a p53-independent fashion (Jeffers et al., 2003; Villunger et al., 2003). The assignment of Puma as either a sensitizer or an activator has been somewhat contentious. The BH3 domains of BH3-only proteins are both necessary and sufficient to interact with Bcl-2 family members and seem to largely recapitulate function of the entire protein. For instance, the BH3 domains of Bid and Bim can activate Bax and Bak in liposomal or mitochondrial settings. The Puma BH3 domain lacked this function in several studies, leading many to classify Puma as a sensitizer (Kuwana et al., 2005; Certo et al., 2006). However, experiments with the full-length protein translated in vitro show an ability to activate Bax comparable with that of Bim and Bid (Kim et al., 2006).Cartron et al. (2004) has previously found that the BH3 domains of Bim and Puma but not the sensitizer Bad interact with Bax and cause its activation. In Gallenne et al. (2009), the role of Puma as an activator is further supported by three main pieces of evidence. First, Bax preincubated with the Puma BH3 peptide is more toxic to microinjected cells than is Bax alone. This enhancement is blocked by coincubation with a peptide mimicking the putative interaction site on Bax, the Hα1 C-terminal peptide. This suggests that the interaction of the Puma BH3 domain with a site on the first α helix of Bax is necessary for Puma''s enhancement of Bax killing. It is worth noting that this interaction site on Bax, first identified by this group 4 yr ago, overlaps with an interaction site of the activator Bim BH3 peptide with Bax recently demonstrated by nuclear magnetic resonance in solution (Gavathiotis et al., 2008). The fact that two groups independently identified a similar and unexpected site for interaction of activating BH3 domains with Bax lends some confidence to this finding.Additionally, because the Bcl-2 family is absent from the yeast genome, the authors exploit yeast to study Puma and Bax in a setting uncontaminated by the contribution of unmeasured Bcl-2 family proteins. Again, they find that coexpression of Puma is necessary for efficient killing by Bax. Finally, the authors investigate the participation of Puma in killing human colorectal cancer cells with ABT-737. ABT-737 is a BH3 mimetic that promotes apoptosis by binding antiapoptotic proteins and displacing select prebound prodeath proteins. Thus, ABT-737 can only kill cells that are primed with either activators or preactivated Bax or Bak. They find that ABT-737 treatment results in the freeing of Puma, which then interacts with Bax, correlating with the death of the cell. This finding suggests that Puma can play the priming function that is likely critical to sensitivity to many chemotherapeutic agents as well as ABT-737 (Deng et al., 2007). This role may be particularly important in cells in which Bim and Bid are not expressed at high levels.Some questions remain. It is not clear why several laboratories have consistently failed to observe an activating function for the BH3 domain of Puma in either mitochondrial or liposomal systems. It is possible that even if Puma can play an activating role, the efficiency of this function may vary considerably according to context and perhaps be much less in many contexts than that of Bid or Bim. In a full-length Puma protein, perhaps interactions of the Puma BH3 domain with Bax are enhanced. It is also possible that unknown posttranslational modifications of Puma or Bax, varying according to cellular context, significantly influence the ability of Puma to activate Bax. In any case, Gallenne et al. (2009) have strengthened the case for Puma as an activator so that its potential contribution to this function cannot be ignored. One must now wonder: what other activators might still be out there waiting to be discovered?     

Mitochondrial Regulation of Cell Death

Although required for life, paradoxically, mitochondria are often essential for initiating apoptotic cell death. Mitochondria regulate caspase activation and cell death through an event termed mitochondrial outer membrane permeabilization (MOMP); this leads to the release of various mitochondrial intermembrane space proteins that activate caspases, resulting in apoptosis. MOMP is often considered a point of no return because it typically leads to cell death, even in the absence of caspase activity. Because of this pivotal role in deciding cell fate, deregulation of MOMP impacts on many diseases and represents a fruitful site for therapeutic intervention. Here we discuss the mechanisms underlying mitochondrial permeabilization and how this key event leads to cell death through caspase-dependent and -independent means. We then proceed to explore how the release of mitochondrial proteins may be regulated following MOMP. Finally, we discuss mechanisms that enable cells sometimes to survive MOMP, allowing them, in essence, to return from the point of no return.In most organisms, mitochondria play an essential role in activating caspase proteases through a pathway termed the mitochondrial or intrinsic pathway of apoptosis. Mitochondria regulate caspase activation by a process called mitochondrial outer membrane permeabilization (MOMP). Selective permeabilization of the mitochondrial outer membrane releases intermembrane space (IMS) proteins that drive robust caspase activity leading to rapid cell death. However, even in the absence of caspase activity, MOMP typically commits a cell to death and is therefore considered a point of no return (Fig. 1). Because of this pivotal role in dictating cell fate, MOMP is highly regulated, mainly through interactions between pro- and antiapoptotic members of the Bcl-2 family. In this article, we begin by discussing how mitochondria may have evolved to become central players in apoptotic cell death. We then provide an overview of current models addressing the mechanics of MOMP, outlining how this crucial event leads to cell death through both caspase-dependent or -independent mechanisms. Finally, we discuss how caspase activity may be regulated post-MOMP and define other processes that allow cells to survive MOMP and, in effect, return from the point of no return.Open in a separate windowFigure 1.Mitochondrial regulation of cell death. Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) can lead to caspase-dependent apoptosis (left) or caspase-independent cell death (right). Following MOMP, soluble proteins are released from the mitochondrial intermembrane space into the cytoplasm. Cytochrome c binds to monomeric Apaf-1 leading to its conformational change and oligomerization. Procaspase-9 is recruited to heptameric Apaf-1 complexes forming the apoptosome. This leads to activation of caspase-9 and, through caspase-9-mediated cleavage, activation of the executioner caspases-3 and -7. Release of Smac and Omi from the mitochondrial intermembrane space facilitates caspase activation by neutralizing the caspase inhibitor XIAP. MOMP can also lead to nonapoptotic cell death through a gradual loss of mitochondrial function and/or release of mitochondrial proteins that kill the cell in a caspase-independent manner.     

The Fowlpox Virus BCL-2 Homologue,FPV039, Interacts with Activated Bax and a Discrete Subset of BH3-Only Proteins To Inhibit Apoptosis

Apoptosis is a potent immune barrier against viral infection, and many viruses, including poxviruses, encode proteins to overcome this defense. Interestingly, the avipoxviruses, which include fowlpox and canarypox virus, are the only poxviruses known to encode proteins with obvious Bcl-2 sequence homology. We previously characterized the fowlpox virus protein FPV039 as a Bcl-2-like antiapoptotic protein that inhibits apoptosis by interacting with and inactivating the proapoptotic cellular protein Bak. However, both Bak and Bax can independently trigger cell death. Thus, to effectively inhibit apoptosis, a number of viruses also inhibit Bax. Here we show that FPV039 inhibited apoptosis induced by Bax overexpression and prevented both the conformational activation of Bax and the subsequent formation of Bax oligomers at the mitochondria, two critical steps in the induction of apoptosis. Additionally, FPV039 interacted with activated Bax in the context of Bax overexpression and virus infection. Importantly, the ability of FPV039 to interact with active Bax and inhibit Bax activity was dependent on the structurally conserved BH3 domain of FPV039, even though this domain possesses little sequence homology to other BH3 domains. FPV039 also inhibited apoptosis induced by the BH3-only proteins, upstream activators of Bak and Bax, despite interacting detectably with only two: BimL and Bik. Collectively, our data suggest that FPV039 inhibits apoptosis by sequestering and inactivating multiple proapoptotic Bcl-2 proteins, including certain BH3-only proteins and both of the critical “gatekeepers” of apoptosis, Bak and Bax.Apoptosis is a highly conserved form of programmed cell death that plays an important role in the immune defense against pathogens. The controlled and deliberate destruction of virally infected cells comprises a potent innate immune barrier against rampant viral replication and infection. As such, many viruses, including poxviruses, encode numerous proteins that inhibit a variety of steps in the biochemical pathways that lead to cell death (29, 69).The mitochondria, and the Bcl-2 family of proteins that preside over them, serve as an important control point in the regulation of apoptosis (87). United by the presence of one to four highly conserved Bcl-2 homology (BH) domains, the Bcl-2 family regulates the integrity of the outer mitochondrial membrane (OMM) and controls the release of apoptogenic molecules from the mitochondrial intermembrane space. Bak and Bax, the two proapoptotic Bcl-2 proteins, possess BH domains 1 to 3 and, upon activation, commit the cell to death (53, 77). Whereas Bak resides constitutively at the OMM, Bax exists in an inactive form in the cytoplasm and, upon apoptotic insult, undergoes a conformational change that exposes its C-terminal transmembrane domain and results in its relocalization to the OMM (10, 34, 41, 56). The attendant exposure of the N termini of both Bak and Bax precedes Bak and Bax homooligomerization, which facilitates mitochondrial damage and, ultimately, the release of cytochrome c (3, 4, 36, 37, 76). Cytochrome c, in turn, triggers the activation of caspases, a group of cysteine proteases responsible for dismantling the apoptotic cell (59). Bak and Bax are therefore crucial for the induction of apoptosis and, because either Bak or Bax alone is sufficient to facilitate the release of cytochrome c, both must be inactivated to effectively inhibit apoptosis (53, 77, 90). The activation of Bak and Bax is counteracted by the antiapoptotic members of the Bcl-2 family, including Bcl-2, Bcl-X_L, and Mcl-1. These three proteins, which possess all four BH domains, reside at the mitochondria and prevent apoptosis by directly interacting with and inhibiting Bak and Bax or the BH3-only proteins (87). The BH3-only proteins, which possess only the BH3 domain, act as sentinels responsive to a variety of cellular stresses, including virus infection (79). Upon receipt of an apoptotic stimulus, BH3-only proteins become activated and subsequently activate Bak and Bax or inhibit the antiapoptotic function of Bcl-2, Bcl-X_L, and Mcl-1 (15). Of the eight BH3-only proteins that are directly involved in the induction of apoptosis—namely, Bim, Bid, Puma, Bik, Bmf, Bad, Noxa, and Hrk—each displays a specific and characteristic ability to bind and inhibit Bcl-2 proteins (79).Like cellular antiapoptotic Bcl-2 proteins, viral inhibitors of apoptosis have evolved especially to interfere with the activation of Bak and Bax (18, 40). For example, E1B 19K, encoded by adenovirus, and M11L, encoded by myxoma virus, bind and inactivate both Bak and Bax to inhibit apoptosis (26, 49, 65, 67, 72). Similarly, ORF125, the antiapoptotic protein encoded by the poxvirus Orf virus, also inactivates Bak and Bax, but exactly how ORF125 mediates this inactivation remains unknown (78). Although interacting with Bak and Bax is ostensibly the most direct way to prevent apoptosis, several viral antiapoptotic proteins appear to inhibit apoptosis by functioning upstream of Bak and Bax at the level of the BH3-only proteins. The vaccinia virus protein F1L, for example, interacts with Bak but not Bax, yet F1L is nonetheless capable of inactivating Bax, likely a result of F1L interacting with the BH3-only protein and Bax activator, Bim (61, 70, 74). Moreover, the Bcl-2 homolog encoded by Kaposi''s sarcoma-associated herpesvirus, and BHRF-1, encoded by Epstein-Barr virus, each interact with a specific and distinct array of BH3-only proteins, yet neither protein interacts detectably with Bak or Bax (14, 27, 44). Thus, to effectively inhibit apoptosis, it may not be necessary for viral proteins to directly target Bak and Bax but, instead, to prevent the activation of Bak and Bax by interfering with the upstream BH3-only proteins (15).Recently, our lab has shown that FPV039, encoded by fowlpox virus, localizes to the mitochondria, where it inhibits apoptosis induced by a variety of stimuli (6). Interestingly, FPV039 is the only characterized poxvirus protein that shares obvious, albeit limited, sequence homology with cellular Bcl-2 proteins (1, 6). FPV039 possesses a highly conserved BH1 and BH2 domain but lacks an obvious BH3 and BH4 domain. Importantly, however, we predicted structural homology between the Bcl-2 BH3 domain and a corresponding region in FPV039, and we validated the prediction by showing that this cryptic FPV039 BH3 domain is functionally important (6). Indeed, the ability of FPV039 to interact with the proapoptotic protein Bak is dependent on this cryptic BH3 domain (6). Thus, despite lacking sequence conservation of a highly conserved BH3 domain, FPV039 is able to interact with, and inactivate, the proapoptotic protein Bak. Nevertheless, to completely inhibit apoptosis, both Bak and Bax must be inactivated.Accordingly, we wanted to determine whether FPV039, in addition to inactivating Bak, could inactivate Bax. We report here that FPV039 inhibited Bax activity and prevented critical steps in Bax activation. FPV039 did not appear to interact with endogenous inactive Bax; however, FPV039 was able to interact with active Bax. Moreover, FPV039 inhibited apoptosis induced by the BH3-only proteins despite interacting with only BimL and Bik. Together, these data strongly suggest FPV039 inhibits apoptosis by inactivating multiple proapoptotic Bcl-2 proteins, including the critical Bak and Bax, as well as a discrete subset of BH3-only proteins.     

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.The regulated elimination of cells by apoptosis is a key mechanism of development, tissue homeostasis and defense. In vertebrates, apoptosis is regulated through two pathways, the death receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway, which is activated by numerous apoptotic stimuli. Mitochondrial apoptosis is characterized by loss of mitochondrial outer membrane integrity and the release of mitochondrial intermembrane space proteins, most notably cytochrome c, which leads to the activation of the caspase-9 and effector caspases.¹Release of cytochrome c is governed by proteins of the B-cell lymphoma 2 (Bcl-2) family.² The Bcl-2 family consists of three groups, whose expression and interaction decide cell survival. The anti-apoptotic Bcl-2 proteins include Bcl-2, Bcl-X_L (B-cell lymphoma-extra large), Bcl-w (Bcl-2-like protein 2), Mcl-1 (myeloid cell leukemia sequence 1) and A1 (Bcl-2-related protein A1). The pro-apoptotic group of BH3-only proteins (containing a BH3-domain: Bim (Bcl-2-interacting mediator of cell death), Bid (BH3-interacting domain death agonist), Puma (p53-upregulated modulator of apoptosis), Noxa (Phorbol-12-myristate-13-acetate-induced protein 1), Bad (Bcl-2-associated death promoter), Bik (Bcl-2-interacting killer) and Hrk (activator of apoptosis hara-kiri)) activate the pro-apoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Bax and Bak can replace each other in most situations, but the presence of one of them is required for mitochondrial apoptosis. Upon activation Bax and Bak form oligomers in the outer mitochondrial membrane and cause the release of cytochrome c. How Bax and Bak are activated is still under debate. Different activation models have been proposed and investigated.According to the direct activation model BH3-only proteins can directly, by physical interaction activate Bax and Bak.³ The model was derived in studies investigating synthetic BH3-domain peptides in in vitro systems, that is, isolated mitochondria or liposomes, where peptides encompassing the BH3-domains of Bim or Bid (‘activator'' BH3-only proteins) were able to activate Bax. Peptides derived from the BH3-only proteins Bad, Bik, Hrk, Noxa or Puma did not activate Bax directly. However, these peptides can bind to anti-apoptotic Bcl-2 proteins with varying preferences.⁴ As this may neutralize a combination of anti-apoptotic proteins it may facilitate Bax/Bak activation by activator BH3-only proteins. Consequently, this group of BH3-only proteins has been named ‘sensitizer'' or ‘derepressor'' BH3-only proteins.^{3, 5, 6, 7} The direct activation model has received recent support by structural studies of activator BH3-domains bound to Bax.⁸ That study also found that the BH3-only peptides used previously lacked a residue that is important in the activation of Bax, and the previous results may have to be reconsidered. Indeed, a recent study illustrates that placing the BH3-domain from the various BH3-only proteins into intact Bid protein enhances Bax/Bak-activating capacity of the BH3-domains of Bid, Bim, Puma, Bmf (Bcl-2-modifying factor), Bik and Hrk.⁹The displacement (or indirect activation) model on the other hand posits that Bax and Bak are held in check by anti-apoptotic Bcl-2 proteins and auto-activate when this interaction is broken by BH3-only proteins (displacement). BH3-only proteins can bind to anti-apoptotic Bcl-2 proteins and upon apoptotic stimulation may cause the displacement of these proteins from Bax and Bak, which may lead to the activation of effectors. BH3-peptides derived from Bim and Puma can bind to all anti-apoptotic Bcl-2 proteins and its corresponding proteins exert killing upon overexpression, whereas Bad, Bmf, Bid, Bik, Hrk and Noxa display binding patterns restricted to certain anti-apoptotic Bcl-2 proteins.⁴ It was therefore suggested that Bax/Bak activation requires the neutralization/displacement of several anti-apoptotic proteins, which may be achieved by one BH3-only protein with broadly binding characteristics (such as Bim) or by the combination of BH3-only proteins with restricted binding capabilities (for instance Bad plus Noxa).^{10, 11}The models have been further refined; the ‘embedded together'' model additionally considers the dynamic interaction of the proteins with the mitochondrial membrane,¹² and it has been proposed that the models can be unified by taking two ‘modes'' of inhibition into account: anti-apoptotic Bcl-2 proteins have a dual function in inactivating both, BH3-only proteins and effectors. Pro-apoptotic signals cause the release of activator BH3-only proteins from sequestration with anti-apoptotic Bcl-2 proteins. Free BH3-only proteins directly activate effectors, however, cell death may still not be initiated because the effectors are then held in check by anti-apoptotic Bcl-2 proteins. Free activator BH3-only proteins are required to activate effectors.¹³This model unifies the two above models in the sense that it incorporates aspects of both, inhibition and displacement as well as direct activation. However, the core difference between the (direct) activation and the displacement model appears to be irreconcilable: in the activation model Bax and Bak are inactive unless receiving a stimulus from BH3-only proteins whereas in the displacement model they are active unless bound to anti-apoptotic proteins. Thus, in the absence of all other proteins one model predicts that Bax/Bak are active, the other that they are inactive. Obviously they cannot be both.The direct activation model has initially been established with Bax and the displacement model with Bak. The data are very strong that Bax is activated by direct interaction with BH3-only proteins. Recombinant Bak can also be directly activated by recombinant tBid,¹⁴ and Bid/BH3-chimaeras can activate recombinant Bak missing its C terminus.⁹ However, since Bak is normally inserted into the outer mitochondrial membrane where it may be bound to numerous other Bcl-2-family members, it has been difficult directly to test activation of Bak in the physiological situation.One possibility to ‘unify'' the original models may be in a model where Bax is physiologically activated by direct activation (Bax is inactive until receiving a signal through BH3-only proteins) whereas Bak is activated indirectly (auto-activates when the inhibition by Bcl-2-like proteins is relieved). Here we test this possibility of indirect Bak activation. We targeted anti-apoptotic Bcl-2 family proteins using RNAi. In this setting, protein concentrations and conditions are physiological, which avoids some of the problems associated with overexpression or cell-free experiments. Non-malignant cells may respond differently to the loss of anti-apoptotic Bcl-2 proteins compared with tumor cells.¹⁵ In this study, using non-malignant cells, we targeted all anti-apoptotic Bcl-2 molecules in combinations of two. In the absence of apoptotic stimuli we observed that the combined loss of Bcl-X_L and Mcl-1 was sufficient to induce apoptosis. The direct activator proteins Bid, Bim and Puma were not needed. These observations provide evidence for indirect activation of Bak.     

Where Killers Meet—Permeabilization of the Outer Mitochondrial Membrane during Apoptosis

Although mitochondria are usually considered as supporters of life, they are also involved in cellular death. Mitochondrial outer membrane permeabilization (MOMP) is a crucial event during apoptosis because it causes the release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. MOMP is mainly controlled by the Bcl-2 family of proteins, which consists of both proapoptotic and antiapoptotic members. We discuss the current understanding of how activating and inhibitory interactions within this family lead to the activation and oligomerization of MOMP effectors Bax and Bak, which result in membrane permeabilization. The order of events leading to MOMP is then highlighted step by step, emphasizing recent discoveries regarding the formation of Bax/Bak pores on the outer mitochondrial membrane. Besides the Bcl-2 proteins, the mitochondrial organelle contributes to and possibly regulates MOMP, because mitochondrial resident proteins and membrane lipids are prominently involved in the process.Mitochondria are essential for the life of the cell. They produce most of the ATP via oxidative phosphorylation thanks to the respiratory chain that is embedded in the inner mitochondrial membrane. Consequently, mitochondrial dysfunction is implicated in the development of many human diseases, in particular, neurodegenerative disorders (Lin and Beal 2006). Mitochondria are also prominently involved in cell death, because they play a crucial role in many apoptotic responses. Apoptosis is a self-destruction program that is essential during the development of multicellular organisms. Its dysregulation has also been recognized as a main feature of many pathological conditions, especially cancer (Llambi and Green 2011).The executioners of apoptosis are a family of cysteine proteases termed caspases that cleave a variety of cellular targets, resulting in morphological changes, degradation of genomic DNA, and, ultimately, phagocytic removal of the apoptotic cell (Taylor et al. 2008). Caspases are synthesized as inactive zymogens that become activated after regulated limited proteolysis. Two different pathways of apoptotic signaling that result in the activation of executioner caspases 3 and 7 can be distinguished. In the extrinsic pathway, binding of ligands such as FasL or TNFα to a death receptor on the plasma membrane leads to the activation of initiator caspase 8. Active caspase 8 propagates the signal by directly cleaving and thereby activating caspases 3 and 7, which continue a proteolytic cascade ultimately leading to the removal of the cell.The intrinsic pathway, on the other hand, is initiated upon exposure to a number of stress situations, including DNA damage. A subclass of the Bcl-2 protein family termed BH3-only proteins (see below) becomes activated after an internal stress stimulus and translocates to the outer mitochondrial membrane (OMM), where they orchestrate a process called mitochondrial outer membrane permeabilization (MOMP). As an outcome of this process, pores are formed in the OMM, membrane integrity is lost, and contents of the intermembrane space gain access to the cytosol. One of the molecules that is rapidly released to the cytosol is cytochrome c, which is normally a soluble electron carrier between respiratory complexes III and IV. Together with the proapoptotic cytosolic factor APAF1, cytochrome c assembles into a caspase-activating complex termed the “apoptosome.” This complex subsequently activates caspase 9, which is able to cleave caspases 3 and 7, proceeding with the same downstream cascade as in the extrinsic pathway. Other intermembrane space proteins also contribute to cell death after being released into the cytosol (e.g., SMAC/Diablo, which blocks the caspase inhibitor protein XIAP).Remarkably, the two pathways are not completely independent. Cross talk between the extrinsic and intrinsic pathways exists because of caspase 8-dependent cleavage of the BH3-only protein Bid. Upon cleavage, Bid becomes activated, and the truncated version, tBid, translocates to the surface of mitochondria to induce MOMP. In so-called type II cells, this mitochondrial feedback loop is needed to induce apoptosis through the extrinsic pathway, because of the requirement of XIAP antagonism by SMAC.The loss of OMM integrity caused by MOMP is usually considered the point of no return in the whole process, because cells are committed to die once MOMP is initiated. Therefore, this process represents a major checkpoint of apoptosis and must be tightly controlled to ensure that it is initiated at the right time and place. The main molecular players of MOMP belong to the Bcl-2 protein family. Integration of proapoptotic and antiapoptotic signals by the network of Bcl-2 proteins determines whether or not the OMM is permeabilized. In the following sections, we describe in detail the stimulatory and inhibitory protein–protein interactions within this family, discussing various models of how the MOMP effectors, Bax and Bak, become activated. Furthermore, we focus on the actual event of membrane permeabilization, summarizing the current understanding of how pores are formed in the OMM by Bax and Bak oligomers.     

BH3 domains other than Bim and Bid can directly activate Bax/Bak

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.     

Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0'' residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.The intrinsic pathway to apoptosis is regulated by interactions between members of three factions of the Bcl-2 protein family: the BH3-only proteins such as Bim and Bid, which initiate the process, the essential effectors Bax and Bak, and the prosurvival members, which oppose the action of both other factions.¹ The interactions between prosurvival Bcl-2 family members and BH3 peptides have been well characterised as the earliest studies with Bcl-x_L and a BakBH3 peptide.² Such complexes are readily formed in solution by incubating the C-terminally (ΔC) truncated prosurvival Bcl-2 protein with a BH3 peptide. The absence of the C-terminal segment that can anchor the Bcl-2 protein in a membrane apparently has little effect on the ensuing complex. That complex is believed to be responsible for the antiapoptotic function of Bcl-2, by sequestration of the BH3 motif either of the so-called BH3-only proteins such as Bim (''mode 1'') or of Bax or Bak (''mode 2'').³Although proapoptotic Bax and Bak have very similar three-dimensional structures to their prosurvival relatives,^{4, 5, 6} until recently^{7, 8} no structure of a complex of either Bax or Bak with a BH3 peptide had been captured, despite an accumulation of evidence that Bax and Bak could be activated directly by interaction with the BH3-only proteins Bid, Bim and possibly others.^{9, 10, 11, 12, 13}Unlike Bak, which is constitutively anchored in the mitochondrial outer membrane (MOM) via its C-terminal segment, Bax is largely cytosolic in healthy cells and accumulates at the MOM only upon a death signal.^{14, 15} There it is believed to display at least two different conformers,^{16, 17} one loosely associated with the MOM and another in which its membrane anchor (helix α9) is inserted into the MOM. In striking contrast to the antiapoptotic relatives of Bcl-2, a construct of Bax lacking its C-terminal membrane anchor, BaxΔC21, has no measurable interaction with BH3 peptides. However, in the presence of the detergent octylglucoside binding is detected by surface plasmon resonance (SPR) for the BH3 peptides of Bim, Bid, Bak and Bax itself with IC50s in the range of 0.1–1μM,^{7, 18} some 100-fold weaker compared with those measured similarly with (for example) Bcl-x_LΔC, where no detergent is required. Weaker interactions between BidBH3 or BimBH3 and BaxΔC as compared with Bcl-x_LΔC are not inconsistent with various models for the function of the Bcl-2 protein family whereby the prosurvival molecules sequester BH3 motifs with high affinity and long half-lives, but proapoptotic Bax and Bak are activated by transient (‘hit-and-run'') interactions with BH3 motifs.^{19, 20, 21}Complexes of BaxΔC21 bound to BH3 peptides from Bid and Bax have been prepared by coincubation of the protein with CHAPS and an excess of the peptides.⁷ Under these conditions, the protein undergoes a conformational change and dimerises via domain swapping of helical segments α2–α5 and α6–α8, dubbed ‘core'' and ‘latch'' domains, respectively. Although this ‘core/latch dimer'' is thought to be an in vitro artefact, its formation is diagnostic for the core and latch separation, which is required for membrane-associated Bax to dimerise via its core domains and then to permeabilise the MOM.⁷ If the latch domain is absent, as in a recombinant construct of GFP fused to Bax α2–α5, the core domain forms BH3:groove symmetric dimers,⁷ which, consistent with a wide body of evidence,^{21, 22, 23, 24, 25} are present in apoptotic pores.Previous work⁷ highlighted the importance of two hydrophobic ‘h0'' residues (Figure 1) in the peptide (I82/I83 in BidBH3) in governing Bid''s ability to activate Bax. Similar to Bid, Bim is also a potent direct activator of Bax, and the ‘h0'' amino acids in Bim are proline and glutamic acid. In the absence of a structure of BimBH3:BaxΔC, it remained unclear how these ‘h0'' residues were accommodated. Here we describe the crystal structures of BimBH3 26- and 20-mer peptides bound to BaxΔC26. Comparison with the structure of BidBH3:BaxΔC21 allows a dissection of the critical contacts between these two peptides and BaxΔC. The binding profiles of mutant BH3 peptides illustrate that BimBH3 binding to Bax is less dependent on the ‘h0'' residues compare with that in the case for BidBH3. The BimBH3 complex displays a similar cavity adjacent to Bax α1, α2, α5 and α8 as seen in the BidBH3 complex. We also describe a structure of BidBH3 bound to a BaxΔC21 mutant, I66A, which is more typical of the BH3 signature of antiapoptotic Bcl-2 family proteins^{7, 26}Open in a separate windowFigure 1BimBH3 binds BaxΔC. (a) BH3 peptide sequences used in this study, indicating the 5 hydrophobic amino-acid positions ‘h0''–‘h4''. (b) The core/latch dimer of BaxΔC26 bound to BimBH3. The two Bax polypeptides, shown here as cartoons, are coloured yellow and grey, and the two Bim peptides cyan and orange. A crystallographic dyad symmetry axis passes through the centre of this particle. (c) Structure of BimBH3:BaxΔC26 complex. The globular unit depicted comprises Bax residues 1–128 from one polypeptide and 129–166 from the other, together with the associated Bim peptide. Bax is represented by its surface and colour coded according to surface charge (blue, positive potential (4kT/e); red, negative potential (−2kT/e); calculated using the Adaptive Poisson–Boltzmann Solver.⁴¹ The trace of the Bim peptide (cyan) is shown with ‘h0'' (P144, E145), ‘h1'' (I148), ‘h2'' (L152), ‘h3'' (I155) and ‘h4'' (F159) represented as sticks. (d) Overlay of BimBH3:BaxΔC26 with BidBH3:BaxΔC21 (PDB:4BD2). Structures represented as cartoon ribbons, yellow for Bax in the Bim complex and magenta for Bax in the Bid complex. The peptides (Bim cyan and Bid blue) stand vertically in the foreground in this view (similar to Figure 1c), with their N termini at the bottom of the figure     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

Vesicular Stomatitis Virus Induces Apoptosis Primarily through Bak Rather than Bax by Inactivating Mcl-1 and Bcl-XL

Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-X_L, which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-X_L. However, inactivation of both Mcl-1 and Bcl-X_L was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-X_L indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.The induction of cell death is a major mechanism by which many viruses cause disease in the tissues they infect (23). In addition, the cytolytic activity of viruses has the potential for therapeutic applications, such as the development of oncolytic viruses for the treatment of cancer (27). Vesicular stomatitis virus (VSV) is well studied as a prototype for negative-strand RNA viruses and is an exceptionally potent inducer of apoptosis in a wide variety of cell types (4, 20, 21). Due to its particularly rapid cytopathic effects, VSV is one of the major viruses being developed as an oncolytic agent (27). VSV is capable of inducing apoptosis by activation of multiple apoptotic pathways. It is important to determine how these pathways are activated and the role that they play in apoptosis induced by VSV in order to understand the virulence and oncolytic activity of the virus, as well as to provide a model to which other viruses can be compared.Previous work showed that wild-type (wt) VSV induces apoptosis via the mitochondrial (intrinsic) pathway through the initiator caspase caspase-9 (4, 19). This is due in part to the inhibition of host gene expression by the VSV M protein (19). The inhibition of host gene expression by M protein is the mechanism by which VSV inhibits the host antiviral response (2, 31) and leads to induction of apoptosis, similar to that induced by pharmacologic inhibitors of host gene expression (19). Additionally, M protein mutants of VSV that are deficient in the ability to inhibit new host gene expression are effective inducers of apoptosis (12, 13, 19, 20). However, in contrast to wt VSV, induction of apoptosis by M protein mutant virus occurs primarily via the extrinsic pathway through the initiator caspase caspase-8 (12, 13). Infection with M protein mutant VSV results in the expression of proapoptotic genes that are suppressed during infection with wt VSV (12). Therefore, in the case of VSV with wt M protein, the induction of apoptosis is most likely mediated by proteins already present in the host cell. Since it has previously been shown that wt VSV activates the intrinsic pathway, we focused on the Bcl-2 family of proteins to determine the role of Bcl-2 family members in apoptosis induced by wt VSV.Bcl-2 family proteins function to either suppress or promote mitochondrial outer membrane permeabilization, thereby regulating the release of proapoptotic factors into the cytosol, such as cytochrome c, apoptosis-inducing factor (AIF), and Smac/Diablo (5). Bcl-2 family proteins are subdivided into three groups, depending on the conservation of Bcl-2 homology (BH) domains and function (reviewed in references 8 and 38). The multidomain antiapoptotic Bcl-2 proteins contain BH domains BH1 to BH4 and function to inhibit apoptosis by binding to proapoptotic Bcl-2 family members. Members of this group include Bcl-2, Bcl-X_L, Mcl-1, Bcl-w, and BFL-1/A1. The proapoptotic Bcl-2 proteins are comprised of two groups, the multidomain proteins and the BH3-only proteins. Bax and Bak are the two main members of the multidomain group, containing BH domains BH1 to BH3. These proteins are primarily responsible for the permeabilization of the mitochondrial outer membrane, if their activity is not suppressed by antiapoptotic Bcl-2 family members. The BH3-only proteins contain only one Bcl-2 homology domain (BH3) and include Bid, Bad, Bim, Puma, Noxa, and Bik, among others. These proteins function as upstream sensors of signaling pathways and convey to other Bcl-2 family proteins the signals to initiate apoptosis. These death signals can be transmitted from the BH3-only proteins by either binding to antiapoptotic proteins, causing the release of Bak and Bax, or binding to Bak and Bax, thereby causing their activation (6).The pathways leading to activation of Bak differ from those that activate Bax. Interestingly, only two antiapoptotic Bcl-2 proteins, Mcl-1 and Bcl-X_L, have been shown to interact with Bak, while Bax appears to be able to interact with all of the antiapoptotic proteins, with the exception of Mcl-1 (7, 35). BH3-only proteins have strong binding affinities to the antiapoptotic proteins, suggesting that their primary role may be to derepress Bak and Bax by binding and inhibiting the antiapoptotic proteins (36). In addition, BH3-only proteins may play a role in activation of Bak and Bax by binding and inducing an activated conformation (6, 34). For some stimuli, such as the protein kinase inhibitor staurosporine (SSP), the topoisomerase II inhibitor etoposide, and UV radiation, Bak and Bax appear to be redundant, in that the deletion of both is required to render cells resistant to these agents (33). In contrast, Bak and Bax were nonredundant in the induction of apoptosis by Neisseria gonorrhoeae and cisplatin, such that both were required for apoptosis to occur (18).In the experiments reported here, the silencing of Bak or Bax expression with small interfering RNA (siRNA) showed that Bak is more important than Bax for the induction of apoptosis in HeLa cells infected with wt VSV. Overexpression of both of the antiapoptotic Bcl-2 family proteins known to interact with Bak, Mcl-1 and Bcl-X_L, delayed the onset of apoptosis, while depletion of Mcl-1 or Bcl-X_L by siRNA transfection prior to infection increased the rate of apoptosis. Furthermore, M protein inhibition of new host gene expression led to the depletion of Mcl-1, enabling the rapid activation of apoptosis. However, inhibition of Bcl-X_L was also required for the initiation of apoptosis, indicating a role for one or more BH3-only proteins. Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, was shown to be important for induction of apoptosis by VSV. Likewise, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.     

Cleavage of Bid by Executioner Caspases Mediates Feed Forward Amplification of Mitochondrial Outer Membrane Permeabilization during Genotoxic Stress-induced Apoptosis in Jurkat Cells

The extent to which the BH3-only protein Bid is important for intrinsic (mitochondria-mediated) apoptotic cell death induced by genotoxic stress remains controversial. In the present study, we examine this issue using a panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably depleted of Bid were far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-induced Bak activation, cytochrome c release, loss of mitochondrial membrane potential, and caspase activation were all decreased in cells lacking Bid. Reconstitution experiments using recombinant proteins and permeabilized Bid-deficient cells demonstrated that truncated Bid (tBid), but not full-length Bid, potently induced Bak activation and the release of cytochrome c. Further, caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as cells overexpressing full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to genotoxic stress. These data suggest that tBid plays an important regulatory role in the execution of DNA damage-induced cytochrome c release and apoptosis. However, the fact that cleavage of Bid to tBid is mediated by executioner caspases suggests that a self-amplifying feed forward loop involving caspases, Bid, and mitochondria may help determine irreversible commitment to apoptosis.Apoptosis is an active form of cell death that plays an essential role during normal embryonic development and in the maintenance of tissue homeostasis in the adult organism (1). Consequently, dysregulation of apoptosis has been implicated as a contributing factor to the onset of different pathological conditions, including cancer. In addition, it is now generally accepted that many genotoxic anticancer drugs are effective against tumor cells for their ability to induce mitochondria-mediated apoptosis (2). Similarly, mutations or the altered expression of pro- and anti-apoptotic proteins can contribute to the development of drug resistance.Execution of apoptosis is mediated by a family of cysteine-dependent aspartate-specific proteases (caspases). During true mitochondria-mediated apoptosis, members of the Bcl-2 family of proteins are the primary regulators of caspase activation for their role in controlling mitochondrial outer membrane permeabilization (MOMP)² (3). The process of MOMP results in the release of cytochrome c, second mitochondria-derived activator of caspase (Smac, also known as DIABLO), and Omi (also known as HtrA2) into the cytosol where they converge to promote the activation of caspase-9 within the apoptotic protease-activating factor-1 (Apaf-1) apoptosome complex. The Bcl-2 family contains proteins with opposing functions, and it is generally thought that the induction of MOMP requires the activation of either Bak or Bax triggered by a Bcl-2 homology 3 (BH3)-only protein (4–6). Indeed, evidence in the literature indicates that cells lacking either Bak or Bax exhibit only subtle defects in MOMP, whereas doubly deficient cells are often found to be highly resistant to mitochondria-mediated apoptosis (7, 8).At present, there are two models for the activation of Bax or Bak by BH3-only proteins. One model argues that BH3-only proteins function as either “sensitizer” (e.g. Bad and Noxa) or “activator” proteins (e.g. truncated Bid (tBid), Bim, and perhaps Puma) (9). In this scenario, a sensitizer protein is needed to displace an activator protein from a prosurvival protein (e.g. Bcl-2, Bcl-x_L, or Mcl-1) to activate Bak or Bax. The second model argues that BH3-only proteins bind and inhibit the function of prosurvival Bcl-2 proteins, which normally bind to and inhibit Bak and Bax (10, 11). Of the seven or so known BH3-only proteins (6), Bid is unique in that it requires post-translational modification for activation, most notably involving caspase-8-mediated cleavage to tBid (12–14). Bid normally resides in the cytosol and possibly the nucleus (15). Upon being cleaved, the C-terminal fragment (tBid) is myristoylated at its newly exposed N terminus, translocates to the outer mitochondrial membrane (OMM), and/or activates Bak or Bax protein (16). Recently, it was shown that the N-terminal cleavage fragment of Bid is quickly ubiquitinated for degradation and that this degradation is necessary for the pro-apoptotic function of tBid (17). The same study also concluded that, although full-length Bid is capable of translocating to the OMM, it is not able to induce MOMP on its own (17). A well characterized example of tBid involvement during apoptosis is in the engagement of the mitochondrial apoptotic pathway in so-called type II cells upon activation of the extrinsic pathway (18).Here, we have investigated whether Bid plays a functional role in the induction of MOMP during apoptosis in response to the genotoxic anticancer drug etoposide. To that end, we used Bax-deficient Jurkat cells that are stably depleted of Bid and evaluated the extent to which these cells underwent drug-induced MOMP. In addition, Jurkat clones in which the intrinsic pathway had been inhibited due to the stable knockdown of Apaf-1 or the overexpression of full-length XIAP or the baculoviral IAP repeats 1 and 2 (BIR1/BIR2) of XIAP were used to gain insight into the molecular requirements necessary for cleavage of Bid to tBid during drug-induced apoptosis. Strikingly, the data showed that etoposide-induced apoptosis was decreased in Bid-deficient Jurkat cells. In particular, cells lacking Bid expression exhibited decreased Bak activation, cytochrome c release, loss of mitochondrial membrane potential (ΔΨ), and caspase activation. Further, incubation of permeabilized Bid-deficient cells with recombinant tBid, but not full-length Bid, induced Bak dimerization and cytochrome c release. Significantly, we also found that cleavage of Bid to tBid occurred strictly downstream of Apaf-1 by a mechanism that required active executioner caspases.     

Evaluation of the BH3-only Protein Puma as a Direct Bak Activator

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as “direct activators” that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (K_D = 26 ± 5 nm) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.     

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax^-/-Bak^-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.     

Vaccinia Virus F1L Interacts with Bak Using Highly Divergent Bcl-2 Homology Domains and Replaces the Function of Mcl-1

The Bcl-2 family regulates induction of apoptosis at the mitochondria. Essential to this regulation are the interactions between Bcl-2 family members, which are mediated by Bcl-2 homology (BH) domains. Vaccinia virus F1L is a unique inhibitor of apoptosis that lacks significant sequence similarity with the Bcl-2 family and does not contain obvious BH domains. Despite this, F1L inhibits cytochrome c release from mitochondria by preventing Bak and Bax activation. Although F1L constitutively interacts with Bak to prevent Bak activation, the precise mechanism of this interaction remains elusive. We have identified highly divergent BH domains in F1L that were verified by the recent crystal structure of F1L (Kvansakul, M., Yang, H., Fairlie, W. D., Czabotar, P. E., Fischer, S. F., Perugini, M. A., Huang, D. C., and Colman, P. M. (2008) Cell Death Differ. 15, 1564–1571). Here we show that F1L required these BH domains to interact with ectopically expressed and endogenous Bak. The interaction between F1L and Bak was conserved across species, and both F1L and the cellular antiapoptotic protein Mcl-1 required the Bak BH3 domain for interaction. Moreover, F1L replaced Mcl-1 during infection, as the Bak·Mcl-1 complex was disrupted during vaccinia virus infection. In contrast to UV irradiation, vaccinia virus infection did not result in rapid degradation of Mcl-1, consistent with our observation that vaccinia virus did not initiate a DNA damage response. Additionally, Mcl-1 expression prevented Bak activation and apoptosis during infection with a proapoptotic vaccinia virus devoid of F1L. Our data suggest that F1L replaces the antiapoptotic activity of Mcl-1 during vaccinia virus infection by interacting with Bak using highly divergent BH domains.     

Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death

During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double null, Ppif^-/-, or Ripk3^-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.     

Synaptic Dysfunction in Neurodevelopmental Disorders Associated with Autism and Intellectual Disabilities

The discovery of the genetic causes of syndromic autism spectrum disorders and intellectual disabilities has greatly informed our understanding of the molecular pathways critical for normal synaptic function. The top-down approaches using human phenotypes and genetics helped identify causative genes and uncovered the broad spectrum of neuropsychiatric features that can result from various mutations in the same gene. Importantly, the human studies unveiled the exquisite sensitivity of cognitive function to precise levels of many diverse proteins. Bottom-up approaches applying molecular, biochemical, and neurophysiological studies to genetic models of these disorders revealed unsuspected pathogenic mechanisms and identified potential therapeutic targets. Moreover, studies in model organisms showed that symptoms of these devastating disorders can be reversed, which brings hope that affected individuals might benefit from interventions even after symptoms set in. Scientists predict that insights gained from studying these rare syndromic disorders will have an impact on the more common nonsyndromic autism and mild cognitive deficits.It is estimated that ∼1% of the human population has an autism spectrum disorder (ASD). ASD has widely varied behavioral manifestations, severity, and comorbid conditions (hence the term “spectrum”), but those diagnosed with autism are characterized by impaired communication and reciprocal social interactions, and restricted and repetitive patterns of activities and interests (Baird et al. 2006). Approximately 70% of those diagnosed with autism also have intellectual disability (ID), and 25% have a seizure disorder (Tuchman and Rapin 2002). There is a strong genetic basis for autism, but the risk architecture is highly heterogeneous, and a large number of genes have been implicated (Abrahams and Geschwind 2008). This daunting phenotypic and etiologic complexity, shared by other major psychiatric illnesses, has slowed progress toward developing new therapies.However, autism researchers are optimistic that the possibility of substantial progress may soon be realized (Krueger and Bear 2011). First, the genes have been discovered for numerous syndromic disorders that prominently feature ASD and ID. Second, these gene mutations have been reproduced in animal models that allow detailed examination of the underlying brain pathophysiology. Third, animal research has converged on altered synaptic function as a likely basis for impaired cognition and possibly ASD. Fourth, insights gained on how synapses function differently in the face of these mutations have suggested novel therapeutic interventions validated in preclinical models and that have shown promise in preliminary human clinical trials. Fifth, the fact that ASD and ID can be diagnosed in early childhood maximizes potential benefits of therapy because it can be started at a time when the brain is most plastic. Finally, animal studies using gene reactivation or pharmacological interventions suggest that substantial improvements can be seen even when treatments begin in adulthood (Ehninger et al. 2008b). Thus, a genetic diagnosis of a developmental brain disorder need not be a “life sentence” of permanent and inexorable mental disability.The path from gene discovery to novel treatment is outlined in Figure 1. This process often begins with astute clinical observations that some patients can be distinguished by a common set of phenotypic traits, thus defining a syndrome. Molecular genetic studies can then be undertaken to test the hypothesis that the syndrome has a genetic cause. In the event that disruption of a single gene or DNA segment causes the disease (i.e., a “highly penetrant” mutation), then it is possible to create an animal model (usually a mouse) that carries the same genetic disruption. Although the effects of the genetic lesion will likely manifest differently at the behavioral level in animals and humans because of differences in the complexity of the brains, it is reasonable to postulate that disruptions in elementary neuronal functions are likely to be shared. Understanding this neuronal pathophysiology is critical for identifying potential therapeutic targets. If these targets can be validated in the animal models, then chemistry ensues to generate molecules that can engage the target and satisfy the pharmacodynamic and pharmacokinetic drug requirements. If they are shown to be safe, drug candidates may then advance to human clinical trials. There are currently clinical trials ongoing in several single-gene syndromic disorders associated with ASD and ID. Most of these target alterations in synaptic signaling.Open in a separate windowFigure 1.The promise of molecular medicine in genetically defined disorders of brain development.The notion that some ASD and associated ID represent “synapsopathies” (or “synaptopathies”) is supported by the preponderance of penetrant mutations in genes associated with synaptic structure and function. The most common single-gene mutations in ASD with ID are associated with fragile X syndrome (FMR1), tuberous sclerosis (TSC1, TSC2), neurofibromatosis (NF1), Angelman syndrome (UBE3A), Rett syndrome (MECP2), the PTEN hamartoma tumor syndrome, and Phelan-McDermid syndrome (SHANK3) (for review, see Betancur 2011). Rare mutations in the neuroligin (NLGN3, NLGN2) and neurexin (NRXN1) genes also cause autism (Jamain et al. 2003). Although this is by no means an exhaustive list of genes implicated in autism (and many await discovery), it is notable nonetheless that these highly penetrant mutations occur in genes that are critical regulators of synaptic function, and further, illuminate biochemical pathways that might be pathogenic in ASD and ID (Fig. 2).Open in a separate windowFigure 2.(A) Schematic of a neuron and axonal-dendritic synapse that depict examples of cellular localization of the various types of defects in ASD/ID. (B) A signaling pathway at the excitatory synapses that couples activity as registered by the release of glutamate to local control of protein synthesis. Disruption of the gene products indicated in the colored boxes greatly increases the risk of ASD/ID. Syndromic disorders with increased prevalence of ASD include Phelan-McDermid Syndrome (SHANK3); Noonan syndrome (RAF1, MEK1); Neurofibromatosis type 1 (NF1); Costello syndrome (H-Ras, MEK1); Cowden syndrome (PTEN); Cardio-facio-cutaneous (CFC) syndrome (MEK1/2); Tuberous sclerosis complex (TSC1/2); Fragile X syndrome (FMRP); Angelman syndrome (AS UBE3a); Rett syndrome (RTT–MeCP2); and Rubinstein-Taybi syndrome (RTS–CREB binding protein, p300). Rare, nonsyndromic ASDs include NLGN3/4 and NRXN1; ID/ASD: SHANK2.In this article, we focus on a few syndromic disorders associated with ASD and ID that are characterized by penetrant mutations in genes that have been shown in animal models to disrupt synaptic function. Our goal is to highlight the similarities and differences in these syndromes and their underlying synaptic pathophysiology. Optimal synaptic function occurs within a narrow dynamic range along many dimensions, and it is not surprising that pathophysiology occurs at the edges of these spectra. What has come as a surprise, however, is that ASD and ID appear to be common consequences of disruptive mutations that cause synaptic pathophysiology at both ends of a spectrum. In other words, both “gain-of-function” and “loss-of-function” mutations can manifest in similar ways. Insights into the pathophysiology of ASD and ID have raised the possibility of therapeutic interventions to bring synapses into a normal operating range.     

Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity.

The evolution of modern cells is arguably the most challenging and important problem the field of biology has ever faced …—Carl R. Woese(Woese 2002)

Current models may accept that all modern eukaryotic cells arose from a single common ancestor (the cenancestral eukaryote), the nature of which is—owing to the lack of direct living or fossil descendants—still highly under debate (de Duve 2007). The chimeric nature of eukaryotic genomes with eubacterial and archaebacterial shares led to a discussion about the origin of this first “proto-eukaryote.” Several models exist (see Fig. 1), which either place the evolution of the nucleus before or after the emergence of the mitochondrion (outlined in Koonin 2010; Martijn and Ettema 2013). According to the different postulated scenarios (summarized in Embley and Martin 2006), eukaryotes in the latter case might have evolved by endosymbiosis between a hydrogen-producing, oxygen-producing, or sulfur-dependent α-proteobacterium and an archaebacterial host (Fig. 1C). The resulting mitochondriate prokaryote would have evolved the nucleus subsequently. In other scenarios (Fig. 1B), the cenancestral eukaryote emerged by cellular fusion or endosymbiosis of a Gram-negative, maybe hydrogen-producing, eubacterium and a methanogenic archaebacterium or eocyte, leading to a primitive but nucleated amitochondrial (archezoan) cell (Embley and Martin 2006, and references therein). As a third alternative, Cavalier-Smith (2002) suggested a common eubacterial ancestor for eukaryotes and archaebacteria (the Neomuran hypothesis) (Fig. 1A).Open in a separate windowFigure 1.Evolution of the last common ancestor of all eukaryotic cells. A schematic depiction of the early eukaryogenesis. Because of the lack of living and fossil descendants, several opposing models are discussed (A–C). The anticipated order of events is shown as a flow chart. For details, see text. (Derived from Embley and Martin 2006; Koonin 2010.)     

The Kinetochore

A critical requirement for mitosis is the distribution of genetic material to the two daughter cells. The central player in this process is the macromolecular kinetochore structure, which binds to both chromosomal DNA and spindle microtubule polymers to direct chromosome alignment and segregation. This review will discuss the key kinetochore activities required for mitotic chromosome segregation, including the recognition of a specific site on each chromosome, kinetochore assembly and the formation of kinetochore–microtubule connections, the generation of force to drive chromosome segregation, and the regulation of kinetochore function to ensure that chromosome segregation occurs with high fidelity.A key objective for cell division is to physically distribute the genomic material to the two new daughter cells. Achieving proper chromosome segregation requires three primary things (Fig. 1): (1) the ability to specifically recognize and detect each unit of DNA; (2) a physical connection between the DNA and other cellular structures to mediate their distribution; and (3) a force-generating mechanism to drive the spatial movement of the DNA to the daughter cells. Although this article focuses on how these processes are achieved during mitosis in eukaryotic cells, these key principles are required for DNA segregation in all organisms, including bacteria. Perhaps the simplest DNA distribution machine is the partitioning system that segregates the small, circular bacterial R1 plasmid (Fig. 1). The R1 partitioning system uses just a single component for each of the three key activities listed above (reviewed in Salje et al. 2010). First, a 160-bp sequence-specific DNA element termed parC allows the partitioning system to recognize a specific region of the plasmid. Second, the DNA-binding protein ParR associates with the parC DNA sequence. ParR can then mediate connections between the plasmid DNA and third factor—the filament forming protein ParM. ParM polymerization is capable of generating force to drive the separation of two replicated copies of the R1 plasmid. The R1 plasmid partitioning system is both simple and elegant, and it demonstrates that it is possible to achieve DNA segregation with only two proteins and a short DNA sequence.Open in a separate windowFigure 1.Core requirements for DNA segregation. Cartoon diagram showing the core activities required for DNA segregation of the bacterial R1 plasmid or eukaryotic chromosomes highlighting the recognition of DNA, physical connections, and force.In striking contrast to the R1 plasmid partitioning system, chromosome segregation in eukaryotes (Fig. 1) requires hundreds of different proteins. Given the ability of the simple R1 partitioning system to efficiently mediate DNA segregation in bacteria, it raises the question of why this added complexity is present in eukaryotes. Importantly, there are significant limitations to the bacterial system that would prevent such a system from working in eukaryotes. For example, bacteria are ∼1–2-µm long, whereas vertebrate cells can be ∼10–50 µm in diameter creating a larger spatial requirement to move the DNA (Fig. 1). In addition, although only a single R1 plasmid is present in each bacterium, human cells have 46 different units of DNA (23 from each parent), which are packaged into chromosomes. Each chromosome must be distributed properly during every cell division. Independently recognizing each of these units to ensure their accurate distribution represents a complex challenge. Indeed, adding even one additional R1 plasmid causes the system to break down, with ParM polymers acting indefinitely, pushing the two most closely positioned units of DNA apart to opposite ends of a cell (Campbell and Mullins 2007). Finally, eukaryotic cells require that chromosome segregation occur with high fidelity to ensure that the two replicated units of DNA are distributed accurately to the two new daughter cells. Even a single chromosome mis-segregation event in a multicellular organism has the potential to lead to lethality, lead to developmental disorders, or contribute to cancer progression (Holland and Cleveland 2009; Gordon et al. 2012), placing a high premium on the accuracy of this process.Despite the differences in complexity between bacterial plasmid partitioning systems and the eukaryotic chromosome segregation machinery, the fundamental requirements for distributing DNA to two new cells are remarkably similar (Fig. 1). First, it is necessary to have a region of each chromosome that is “recognized” by the chromosome segregation machinery. In eukaryotes, this region of DNA is termed the centromere. Second, a group of proteins must assemble on this DNA element to facilitate its “connections” to other structures in the cell. In eukaryotes, this physical connection is provided by a macromolecular structure termed the kinetochore. The kinetochore is an impressive molecular machine that requires the coordinated functions of more than 100 different protein components (Cheeseman and Desai 2008). Third, the kinetochore must interact with additional structures that provide the “force” to move the chromosomes. Chromosome segregation in eukaryotes requires microtubule polymers that generate force primarily through their depolymerization.In this review, I will discuss the molecular mechanisms that underlie kinetochore function, including the recognition of a specific site on each chromosome, the formation of the physical kinetochore–microtubule connections, and the forces that drive chromosome segregation during mitosis in eukaryotes, as well as the mechanisms that regulate kinetochore function.