[Show abstract][Hide abstract] ABSTRACT: Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.
PLoS ONE 06/2012; 7(6):e37782. DOI:10.1371/journal.pone.0037782 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.
[Show abstract][Hide abstract] ABSTRACT: The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.
Cancer Research 12/2011; 72(3):736-46. DOI:10.1158/0008-5472.CAN-11-2584 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute exposure to ionizing radiation can cause lethal damage to the gastrointestinal (GI) tract, a condition called the GI syndrome. Whether the target cells affected by radiation to cause the GI syndrome are derived from the epithelium or endothelium and whether the target cells die by apoptosis or other mechanisms are controversial issues. Studying mouse models, we found that selective deletion of the proapoptotic genes Bak1 and Bax from the GI epithelium or from endothelial cells did not protect mice from developing the GI syndrome after sub-total-body gamma irradiation. In contrast, selective deletion of p53 from the GI epithelium, but not from endothelial cells, sensitized irradiated mice to the GI syndrome. Transgenic mice overexpressing p53 in all tissues were protected from the GI syndrome after irradiation. These results suggest that the GI syndrome is caused by the death of GI epithelial cells and that these epithelial cells die by a mechanism that is regulated by p53 but independent of apoptosis.
[Show abstract][Hide abstract] ABSTRACT: The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.
[Show abstract][Hide abstract] ABSTRACT: The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.
Nature medicine 03/2008; 14(2):144-53. DOI:10.1038/nm1717 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.
[Show abstract][Hide abstract] ABSTRACT: Soft tissue sarcomas are mesenchymal tumors that are fatal in approximately one-third of patients. To explore mechanisms of sarcoma pathogenesis, we have generated a mouse model of soft tissue sarcoma. Intramuscular delivery of an adenovirus expressing Cre recombinase in mice with conditional mutations in Kras and Trp53 was sufficient to initiate high-grade sarcomas with myofibroblastic differentiation. Like human sarcomas, these tumors show a predilection for lung rather than lymph node metastasis. Using this model, we showed that a prototype handheld imaging device can identify residual tumor during intraoperative molecular imaging. Deletion of the Ink4a-Arf locus (Cdkn2a), but not Bak1 and Bax, could substitute for mutation of Trp53 in this model. Deletion of Bak1 and Bax, however, was able to substitute for mutation of Trp53 in the development of sinonasal adenocarcinoma. Therefore, the intrinsic pathway of apoptosis seems sufficient to mediate p53 tumor suppression in an epithelial cancer, but not in this model of soft tissue sarcoma.
Nature Medicine 09/2007; 13(8):992-7. DOI:10.1038/nm1602 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c (CYT c) is a protein that employs the caspase recruitment domain (CARD)-containing proteins APAF-1 and CASP-9 to activate effectors CASP-3 and -7. By using affinity labeling techniques and mass spectrometry analysis, we show that histone H1.2 is a regulator of caspases upon UV irradiation. We demonstrated that histone H1.2 forms a protein complex with APAF-1, CASP-9 and CYT c upon UV irradiation. In cell-free systems, we show that histone H1.2 triggers activation of CASP-3 and -7 via APAF-1 and CASP-9. We therefore conclude that upon DNA damage histone H1.2 acts as a positive regulator of apoptosome formation.
[Show abstract][Hide abstract] ABSTRACT: The p53-hDM2 protein interaction is a validated therapeutic target in cancer. We report the synthesis of stabilized alpha-helix of p53 (SAH-p53) compounds that antagonize the p53-hDM2 interaction. We demonstrate that hydrocarbon stapling confers cellular permeability to a p53 peptide that is then capable of modulating transcriptional activity. The lead SAH-p53 compound triggers apoptosis in hDM2-overexpressing cancer cells by reactivating the native p53 signaling pathway. SAH-p53 is the first example of an all-hydrocarbon i, i+7 stabilized peptide that subverts cancer through direct modulation of a transcriptional pathway.
Journal of the American Chemical Society 04/2007; 129(9):2456-7. DOI:10.1021/ja0693587 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Originally discovered as epigenetic regulators of developmental gene expression, the Polycomb (PcG) and trithorax (trxG) group of proteins form distinct nuclear complexes governing post-translational modification of histone tails. This study identified a novel, developmentally regulated interface between Eed and Mll, pivotal constituents of PcG and trxG pathways, respectively, in mouse brain. Although the PcG proteins Eed and EzH2 (Enhancer of Zeste protein-2) engaged in a common complex during neurodevelopment, Eed associated with the trxG protein Mll upon brain maturation. Comprehensive analysis of multiple histone modifications revealed differential substrate specificity of the novel Eed-Mll complex in adult brain compared with the developmental Eed-EzH2 complex. Newborn brain from eed heterozygotes and eed;Mll double heterozygotes exhibited decreased trimethylation at lysine 27 of histone H3, as well as hyperacetylation of histone H4. In contrast, adult hippocampus from Mll heterozygotes was remarkable for decreased acetylation of histone H4, which restored to wild-type levels in eed;Mll double heterozygotes. A physiological role for the Eed-Mll complex in adult brain was evident from complementary defects in synaptic plasticity in eed and Mll mutant hippocampi. These results support the notion that developmental regulation of complex composition bestows the predominant Eed complex with the chromatin remodeling activity conducive for gene regulation during neurodevelopment and adult brain function. Thus, this study suggests dynamic regulation of chromatin complex composition as a molecular mechanism to co-opt constituents of developmental pathways into the regulation of neuronal memory formation in adult brain.
[Show abstract][Hide abstract] ABSTRACT: The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed
cell death. Among the “BCL-2 homology (BH) 3-only” members of pro-apoptotic proteins, truncated BID (tBID) has been implicated
in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 peptide alone at
submicromolar concentrations cannot activate BAX or complement BID BH3 mutant-tBID in mitochondrial and liposomal release
assays. Because tBID contains structurally defined membrane association domains, we investigated whether membrane targeting
of BID BH3 peptide would be sufficient to restore its pro-apoptotic activity. We developed a Ni2+-nitrilotriacetic acid liposomal assay system that efficiently conjugates histidine-tagged peptides to a simulated outer mitochondrial
membrane surface. Strikingly, nanomolar concentrations of a synthetic BID BH3 peptide that is chemically tethered to the liposomal
membrane activated BAX almost as efficiently as tBID itself. These results highlight the importance of membrane targeting
of the BID BH3 domain in tBID-mediated BAX activation and support a model in which tBID engages BAX to trigger its pro-apoptotic
[Show abstract][Hide abstract] ABSTRACT: BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.
[Show abstract][Hide abstract] ABSTRACT: TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.
[Show abstract][Hide abstract] ABSTRACT: Taspase1 was identified as the threonine endopeptidase that cleaves mixed-lineage leukemia (MLL) for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1(-/-) mice. Taspase1 deficiency results in noncleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in the cell cycle. Taspase1(-/-) animals are smaller in size. Taspase1(-/-) mouse embryonic fibroblasts (MEFs) exhibit impaired proliferation, and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down-regulation of Cyclin Es, As, and Bs and up-regulation of p16(Ink4a) . We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The uncleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, chromatin immunoprecipitation assays demonstrate a markedly decreased histone H3 K4 trimethylation at Cyclin E1 and E2 genes in Taspase1(-/-) cells. Furthermore, MLL(nc/nc;2nc/nc) MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1(-/-) cells are resistant to oncogenic transformation, and Taspase1 is overexpressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.
Genes & Development 10/2006; 20(17):2397-409. DOI:10.1101/gad.1449406 · 10.80 Impact Factor