A central issue in the regulation of apoptosis by the Bcl-2 family is whether its BH3-only members initiate apoptosis by directly binding to the essential cell-death mediators Bax and Bak, or whether they can act indirectly, by engaging their pro-survival Bcl-2-like relatives. Contrary to the direct-activation model, we show that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. Our results indicate that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak.
Macrophages play a key role in both normal and pathological processes involving immune and inf lammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor  (TGF-) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1, tumor necrosis factor ␣ (TNF-␣), interleukin 2, and macrophage colony-stimulating factor but not interferon ␥, or lipopolysaccharide (LPS). Its expression is also increased by TGF-. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of M r 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-␣ production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinf lammatory cytokines and TGF- may serve to limit the later phases of macrophage activation.
In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.
Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-beta receptor II, extracellular signal-regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.
Apoptosis is held in check by prosurvival proteins of the Bcl-2 family. The distantly related BH3-only proteins bind to and antagonize them, thereby promoting apoptosis. Whereas binding of the BH3-only protein Noxa to prosurvival Mcl-1 induces Mcl-1 degradation by the proteasome, binding of another BH3-only ligand, Bim, elevates Mcl-1 protein levels. We compared the threedimensional structures of the complexes formed between BH3 peptides of both Bim and Noxa, and we show that a discrete C-terminal sequence of the Noxa BH3 is necessary to instigate Mcl-1 degradation.apoptosis ͉ Bim ͉ Noxa ͉ crystallography T he mammalian Bcl-2-related antiapoptotic proteins (Bcl-2, Bcl-x L , Bcl-w, Mcl-1, and A1) are critical for maintaining cell survival during development or in response to various stress stimuli (1). They share up to four Bcl-2 homology domains, BH1 through BH4, and contain a putative membrane anchoring sequence at their C termini. Structural studies on proteins lacking only this C-terminal segment reveal that the Bcl-2 family fold is that of an all-helical protein in which the BH1, BH2, and BH3 domains are spatially clustered around a depression on the protein surface (2-5). In response to death signals, such as cytotoxic agents or radiation, a related protein family (the BH3-only proteins) antagonizes the function of the antiapoptotic proteins. The BH3 domains of these proapoptotic molecules form an amphipathic ␣-helical fold when bound to a groove lined by the BH1, BH2, and BH3 domains of antiapoptotic proteins such as Bcl-x L (6-8), a step thought to be important for apoptosis induction.Mcl-1 (myeloid cell factor 1) (9) has features distinguishing it from the other prosurvival proteins. It has a central and nonredundant role in the maintenance of progenitor and stem cells (10-12). The levels of Mcl-1 are highly regulated. In some cell types, signals for differentiation trigger its up-regulation (13), whereas basal levels are controlled, at least in part, by the ubiquitin-proteasome machinery. The HECT domain-containing E3 ligase Mule controls basal Mcl-1 protein abundance and induces its degradation in response to DNA-damaging agents such as cisplatin (14). Mule harbors a BH3 domain that allows it to bind Mcl-1. Noxa is a BH3-only protein that can bind and trigger proteasome-mediated Mcl-1 degradation (15). Whether Mule and Noxa contribute to the proteasomal degradation of Mcl-1 in response to UV irradiation (16) or viral infection is unclear (17). Furthermore, the structural basis for Mcl-1 degradation induced by Noxa is unknown.Recently we have shown that the five mammalian antiapoptotic molecules cluster into two classes; one (containing Bcl-2, Bcl-x L , and Bcl-w) is neutralized by the BH3-only protein Bad, and the other (containing Mcl-1 and A1) is neutralized by the BH3-only protein Noxa (18). Inactivation of both subsets of prosurvival proteins appears necessary for cell death to proceed. Interestingly, the recently described Bcl-2 antagonist ABT-737 (19) is a Bad-like BH3 mimetic and does not bind M...
Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-xL and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-xL, Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.
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