The murine septin4 gene (Sept4) has been implicated in diverse cellular functions, including cytokinesis, apoptosis, and tumor suppression. Here, we investigated the function of Sept4 proteins during mouse development by creating a targeted deletion of the Sept4 genomic locus. Sept4 mutant mice are viable but male sterile due to immotile and structurally defective sperm. During spermatogenesis, Sept4 proteins were essential for proper mitochondrial architecture and establishment of the annulus, a ring-like structure in the tail region of sperm. In addition, Sept4 mutant sperm showed defects in the elimination of residual cytoplasm during sperm maturation and had increased staining for the caspase inhibitor XIAP. This is consistent with a role of the proapoptotic Sept4 protein ARTS in promoting caspase-mediated removal of cytoplasm via inhibition of XIAP. Our results indicate that Sept4 proteins play distinct but evolutionarily conserved functions in different cellular compartments.

Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-beta signalling pathway). ARTS is expressed in many cells and acts to enhance cell death induced by TGF-beta or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.

ARTS is an unusual septin-like mitochondrial protein that was originally shown to mediate TGF-beta-induced apoptosis. Recently, we found that ARTS is also important for cell killing by other pro-apoptotic factors, such as arabinoside, etoposide, staurosporine and Fas. In Drosophila, the IAP antagonists Reaper, Hid and Grim are essential for the induction of virtually all apoptotic cell death. We found that mutations in peanut, which encodes a Drosophila homologue of ARTS, can dominantly suppress cell killing by Reaper, Hid and Grim, indicating that peanut acts downstream or in parallel to these. In mammalian cells, ARTS is released from mitochondria upon pro-apoptotic stimuli and then binds to XIAP. Binding of ARTS to XIAP is direct, as recombinant ARTS and XIAP proteins can bind to each other in vitro. ARTS binding to XIAP is specific and related to its pro-apoptotic function, as mutant forms of ARTS (or related septins) that fail to bind XIAP failed to induce apoptosis. ARTS leads to decreased XIAP protein levels and caspase activation. Our data suggest that ARTS induces apoptosis by antagonizing IAPs.

Inhibitor of Apoptosis Proteins (IAPs) are frequently overexpressed in tumors and have become promising targets for developing anti-cancer drugs. IAPs can be inhibited by natural antagonists, but a physiological requirement of mammalian IAP antagonists remains to be established. Here we show that deletion of the mouse Sept4 gene, which encodes the IAP antagonist ARTS, promotes tumor development. Sept4-null mice have increased numbers of hematopoietic stem and progenitor cells, elevated XIAP protein, increased resistance to cell death, and accelerated tumor development in an Em-Myc background. These phenotypes are partially suppressed by inactivation of XIAP. Our results suggest that apoptosis plays an important role as a frontline defense against cancer by restricting the number of normal stem cells.[Keywords: Apoptosis; cancer; tumor suppressor; IAP; stem cells; lymphoma] Supplemental material is available at http://www.genesdev.org.

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis. Apoptosis is important for regulating cell numbers and maintaining tissue homeostasis. The main executioners of apoptosis are caspases, a family of cysteine proteases that cleave substrates after aspartate. 1 In the mitochondrial pathway, release of pro-apoptotic factors, including cytochrome C (cytoC) and Smac/Diablo (SMAC), from the mitochondrial intermembrane space (IMS) to the cytosol promotes caspase activation. This release requires mitochondrial outer membrane permeabilization (MOMP). 2 A holoenzyme complex known as the 'apoptosome' is formed when cytoC is released from mitochondria and binds to apoptotic protease-related factor-1 (Apaf-1) to activate procaspase-9. 3 The best-studied family of caspase inhibitors is the inhibitor of apoptosis (IAP) proteins. 4 IAP proteins contain at least one baculoviral IAP repeat (BIR) domain which can directly interact with caspases and inhibit their apoptotic activity, a RING domain that bestows E3-ubiquitin ligase activity and an Ubiquitin-associated (UBA) domain, which enables the binding of polyubiquitin conjugates via lysine 63. 5-7 X-linked IAP (XIAP) directly inhibits caspases-3, -7 and -9. 8 XIAP is considered to be the most potent inhibitor of caspases in vitro, and elevated levels of this protein are found in human cancers. 9 Although XIAP-null mice are viable, it was recently shown that loss of XIAP function causes elevated caspase-3 activity and sensitizes certain primary cells toward apoptosis. 7 In dying cells, apoptosis can be overcome through the release of caspases from their binding to IAP proteins. [10][11][12][13] Several mammalian XIAP antagonists have been identified, including SMAC, 14,15 Omi/HtrA2 16 and ARTS. 17,18 SMAC and Omi/HtrA2 are located in the mitochondrial IMS, contain a conserved IAP-binding motif (IBM) and are released to the cytosol upon apoptotic induction. 14,15 Genetic inactivation of SMAC and Omi/HtrA2 has fail...

Summary We describe a mechanism by which the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein is down-regulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X linked Inhibitor of Apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP and Sept4/ARTS deficient MEFs, demonstrating that XIAP serves as an E3-ligase for Bcl-2 and ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.

Acquired resistance towards apoptosis is the hallmark of most if not all types of cancer. We have previously identified and characterized ARTS, a broadly expressed protein localized to mitochondria. ARTS was initially shown to mediate TGF-b induced apoptosis. Recently, we have found that high levels of ARTS induce apoptosis without additional pro-apoptotic stimuli. Further, ARTS promotes apoptosis in response to a wide variety of proapoptotic stimuli. Here, we report that the expression of ARTS is lost in all lymphoblasts of more than 70% of childhood acute lymphoblastic leukemia (ALL) patients. The loss of ARTS is specific, as the related non-apoptotic protein H5, bearing 83% identity to ARTS, is unaffected. During remission, ARTS expression is detected again in almost all patients. Two leukemic cell lines, ALL-1 and HL-60 lacking ARTS, were resistant to apoptotic induction by ara-C. Transfection of ARTS into these cells restored their ability to undergo apoptosis in response to this chemotherapeutic agent. We found that methylation process contributes to the loss of ARTS expression. We conclude that the loss of ARTS may provide a selective advantage for cells to escape apoptosis thereby contributing to their transformation to malignant lymphoblasts. We therefore propose that ARTS can function as a tumor suppressor protein in childhood ALL.

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