Life in the balance: how BH3-only proteins induce apoptosis
Introduction
The cell suicide program termed apoptosis removes damaged, infected and superfluous cells. In most circumstances, a cell's decision whether to live or die rests largely with the Bcl-2 family of interacting proteins [1•, 2•]. In mammalian cells, its pro-survival members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1) oppose two pro-apoptotic groups: the Bax group and the BH3-only proteins. Members of the Bax group (Bax, Bak and Bok) are structurally similar to Bcl-2 and bear three ‘BH’ (Bcl-2 homology) domains, whereas the BH3-only proteins, which include Bim, Bad, Bid, Bik, Bmf, Puma, Noxa and Hrk, share only the BH3 interaction domain. The BH3-only proteins monitor cellular wellbeing and, when activated by cytotoxic signals, engage pro-survival relatives by inserting the BH3 domain, an amphipathic α helix, into a hydrophobic groove on their surface. This primes the cell for apoptosis, but commitment also requires activation of Bax or Bak [3, 4]. Once activated, Bax and Bak form oligomers in intracellular membranes, including the mitochondrial outer membrane. The resulting membrane permeabilization releases pro-apoptotic proteins, such as cytochrome c, that provoke activation of the caspases mediating cell demolition [5].
As reviewed previously [1•, 2•, 6, 7, 8, 9•, 10], the BH3-only proteins participate in vital biological processes, and their absence contributes to autoimmunity and neoplasia. Here we review recent advances on their regulation and biological roles. Pertinent to cancer therapy, it has become evident that various BH3-only proteins mediate the cytotoxic responses elicited by chemotherapeutic agents, and there is exciting progress towards creating ‘BH3 mimetics’ as novel anti-cancer agents [11••, 12••]. The recent discovery of selective association of BH3-only proteins with certain pro-survival relatives has delineated functional subclasses of the pro-survival proteins [13••, 14••]. A new function for BH3-only proteins has emerged with discoveries that Noxa [14••] and the novel BH3-bearing ubiquitin ligase Mule/ARF-BP1 [15••] promote Mcl-1 degradation. Finally, we address the vexed issue of whether BH3-only proteins trigger activation of Bax and Bak directly [16, 17•, 18••] or whether they do so indirectly by inactivating the pro-survival family members that guard Bax and Bak [14••].
Section snippets
BH3-only proteins provide fine control over apoptotic responses
The multiplicity and complex regulation of mammalian BH3-only proteins allows exquisite control over apoptosis. Studies with cells from knockout mice indicate that one or a few BH3-only proteins have a dominant role in the responses to diverse cytotoxic stimuli (Figure 1). They mediate death induced not only by physiological stimuli such as cytokine deprivation or detachment from the cell matrix (a type of cell death termed anoikis) but also by the signals induced by activated oncogenes, DNA
Diverse modes of regulation
To avoid unwarranted cell death, BH3-only proteins are restrained by multiple mechanisms [1•, 2•, 7]. Hrk/DP5, Bim, Puma and Noxa are subject to transcriptional control. As reviewed elsewhere [1•, 2•, 7], post-transcriptional constraints include sequestration of phosphorylated Bad by 14-3-3 proteins and production of Bid as a largely inert protein requiring cleavage by, for example, caspase-8 to generate the active truncated product, tBid (Figure 1). In some cell types, both Bim and Bmf seem to
BH3-only proteins selectively engage Bcl-2 pro-survival proteins
The interaction of BH3-only proteins with pro-survival relatives has been most clearly visualized in the high-resolution 3D structure of a Bim BH3 fragment bound to Bcl-xL [35••]. Four hydrophobic pockets in the Bcl-xL groove accommodate the four conserved hydrophobic BH3 residues on one face of the BH3 ligand, whereas hydrophilic residues along the groove make salt bridges with the hydrophilic face of the BH3. Since the bound Bim BH3 α helix spanned at least 29 residues, with extensive groove
Efficient apoptosis requires neutralization of two subsets of pro-survival proteins
The selectivity of BH3-only proteins for their pro-survival counterparts markedly affects their ability to kill cells (Figure 2). Bim and Puma, which target all pro-survival proteins, are more potent killers than BH3-only proteins that target only a subset [13••]. The complementary binding profiles of Bad and Noxa (X) suggests that apoptosis might require neutralization of two classes of Bcl-2 pro-survival proteins, one comprising Bcl-2, Bcl-xL and Bcl-w and the other Mcl-1 and A-1. Notably,
Mcl-1 and Bcl-xL sequester Bak in healthy cells
Analysis of Bak regulation revealed why efficient killing requires neutralization of two classes of pro-survival proteins [14••]. Mcl-1 had been implicated in Bak regulation by its formation of complexes with Bak [38•] and its degradation early in apoptosis [39•]. However, targeting Mcl-1 via either RNAi [39•] or Noxa overexpression failed to kill cells [13••], implicating additional pro-survival relatives. Indeed, in healthy cells Bak was bound by both Mcl-1 and Bcl-xL (Figure 3) but,
Noxa and Mule promote Mcl-1 degradation
Intriguingly, Noxa binding to Mcl-1 prompts proteasome-dependent degradation of Mcl-1 [14••]. However, Mcl-1 poly-ubiquitination can be catalyzed by the novel E3 ubiquitin ligase Mule/ARF-BP1, which also specifically binds Mcl-1 via a Bak-like BH3 domain [15••]. Hence, there may be two routes to Mcl-1 degradation, with the Noxa-induced degradation involving another ubiquitin ligase, presumably one lacking a BH3 domain. The respective roles of Noxa and Mule in the constitutive turnover of Mcl-1
Models for the activation of Bak and Bax
Although BH3-only proteins require either Bax or Bak for cell killing [3, 4], it remains unresolved whether they control the activation of Bax and Bak directly or indirectly [10].
The structural similarity of Bax to pro-survival proteins [35••, 40] has encouraged the view that certain BH3-only proteins directly engage Bax. Letai et al. [16] proposed that BH3-only proteins comprised both ‘sensitisers’, which only inactivate the pro-survival proteins, and ‘activators’, which directly engage Bax
Physiological and pathological functions of BH3-only proteins
As reviewed further elsewhere [9•], gene disruption is clarifying the physiological functions of BH3-only proteins. Bim-deficient mice have been particularly informative. They revealed that Bim is critical for removing superfluous hematopoietic cells, for apoptosis induced by cytokine deprivation and for prevention of autoimmunity [19]. Bim is crucial not only for eliminating lymphocytes that recognize self-antigens, during the development of both T cells [45] and B cells [46], but also for
Roles of BH3-only proteins in tumorigenesis
The apoptosis provoked by DNA damage requires p53 and is considered central to p53 tumor suppression function [57]. Two of its transcriptional targets encode Puma and Noxa, which appear to mediate its apoptotic function. Indeed, Puma-deficient lymphocytes proved nearly as refractory to DNA damage as those lacking p53 [20••, 21••] whereas Noxa has a more restricted role in fibroblasts [20••, 22]. Interestingly, the expression of Noxa, Puma, Bim and Hrk/DP5 is also directly upregulated by the
Implications of BH3-only proteins for cytotoxic therapy
Many anti-cancer agents act through specific BH3-only proteins (Figure 1). For example, histone deacetylase (HDAC) inhibitors, which are showing promise in killing cancer cells, may act by upregulating bmf transcription [65]. Similarly, proteasome inhibitors like bortezomib (Velcade), now in several clinical trials, appear to work by upregulating various BH3-only proteins [66•, 67, 68, 69]. Thus, bortezomib kills melanoma cells but, surprisingly, not normal melanocytes, and acts in part by
The promise of BH3 mimetic anti-cancer drugs
The appeal of BH3 mimetics as a new class of anti-cancer agent [71] is that most tumors have defects in the p53 pathway, precluding induction of the BH3-only proteins Noxa and Puma, and many over-express Bcl-2. A drug that directly targeted pro-survival proteins might therefore be effective. Two approaches to the creation of BH3 mimetics are now showing promise.
One approach uses a modified BH3 peptide [11••]. A 24-mer Bid BH3 peptide, constrained by ‘hydrocarbon stapling’ between two residues
Conclusions
As the BH3-only proteins have now been implicated in many cytotoxic responses (Figure 1), we surmise that nearly all such signals are funneled through one or more of them. The discovery that Bim, Puma and tBid engage all the pro-survival proteins but that the other death ligands engage only subsets (Figure 2) is helping to resolve how these death ligands flip the apoptotic switch. In the activation of Bak, the BH3-only proteins appear to act indirectly by displacing Bak from its sole guards,
Update
A non-apoptotic role has now been ascribed to full-length Bid [72, 73], and Puma reportedly linked to a putative apoptotic role for cytoplasmic p53 [74].
Intact Bid is thought to be largely inert, but two studies now suggest that it has a non-apoptotic role in the DNA damage response [72, 73]. Both conclude that, upon DNA damage, Bid is phosphorylated by the ATM kinase and is needed for S-phase growth arrest. This role does not require its BH3 domain and promotes cell survival, but,
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank our colleagues David Huang, Andreas Strasser, Suzanne Cory, Peter Colman, Mark Hinds, Alan Harris, Philippe Bouillet, Ruth Kluck and Hamsa Puthalakath for valuable discussions. Our research is supported by a NHMRC Program grant (257502), a Specialized Center of Research grant from the Leukemia and Lymphoma Society, and NIH project grants CA80188 and CA43540. SNW holds a fellowship from the Cancer Council of Victoria.
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