Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity

Beclin 1, identified as a Bcl-2-interacting protein, is known to enhance autophagy. However, the effect of Beclin 1 on apoptotic signaling has remained unclear. Here, we show that overexpression of Beclin 1 in MKN28 human gastric cancer cells augmented cis-diamminedichloroplatinum (CDDP)-induced apoptosis. Conversely, "knockdown" of Beclin 1 by a small inhibitory RNA in MKN 1 cells attenuated this cytotoxicity. Furthermore, not only caspase-3/7 activities, but also caspase-9 activity was increased in Beclin 1 gene transfectants treated with CDDP, and caspase-9 inhibitor completely abolished augmentation of CDDP-induced apoptosis by Beclin 1 as did a caspase-3 inhibitor. Thus, Beclin 1 augments CDDP-induced apoptosis through enhancing caspase-9 activity and functions as a pro-apoptotic molecule.     

Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays anessential role in autophagy. However, the molecular mechanism by which Beclin 1functions remains largely unknown. Here we report the crystal structure of theevolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Åresolution. Beclin 1 ECD exhibits a previously unreported fold, with threestructural repeats arranged symmetrically around a central axis. Beclin 1 ECDdefines a novel class of membrane-binding domain, with a strong preference forlipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1ECD, comprising three aromatic amino acids, acts as a hydrophobic finger toassociate with lipid membrane, consequently resulting in the deformation ofmembrane and liposomes. Mutation of these aromatic residues rendered Beclin 1unable to stably associate with lipid membrane in vitro and unable tofully rescue autophagy in Beclin 1-knockdown cells in vivo. Theseobservations form an important framework for deciphering the biologicalfunctions of Beclin 1.     

氨基葡萄糖硫酸盐诱导凋亡中Cathepsin D与Bcl-xL的细胞内共定位研究

目的:探讨氨基葡萄糖硫酸盐(Glucosamine Sulfate,GS)诱导K562细胞凋亡中溶酶体Cathepsin D释放后与Bcl-xL的细胞内共定位关系。方法:采用5.0mmol.L-1 GS诱导K562细胞凋亡,通过HE染色和流式细胞仪检测细胞凋亡,应用免疫荧光染色结合激光共聚焦显微镜观察5.0mmol.L-1 GS诱导凋亡前后Cathepsin D与Bcl-xL的细胞内共定位关系。结果:GS诱导K562细胞72小时后出现细胞凋亡的形态改变,流式细胞仪检测表明细胞出现凋亡,诱导凋亡后的Cathepsin D与Bcl-xL的细胞内共定位信号增强。结论:GS诱导K562细胞凋亡后Cathepsin D与Bcl-xL存在细胞内共定位关系,具备一定相互作用的空间基础。     

Structural insights of the MenD from Escherichia coli reveal ThDP affinity

MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate) synthase belongs to the superfamily of thiamin diphosphate-dependent decarboxylases, which converts isochorismate and 2-oxoglutarate to SHCHC, pyruvate, and carbon dioxide. Here, we report the first crystal structure of apo-MenD from Escherichia coli determined in tetragonal crystal form. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes. Analytical gel filtration shows that EcMenD behaves as a dimer as well as a tetramer. Circular dichroism and isothermal calorimetry results confirm EcMenD dependency on ThDP, which concomitantly helps to stabilize with better configuration.     

The Beclin 1 network regulates autophagy and apoptosis

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, a process of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle. It interacts with several cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, nPIST, VMP1, SLAM, IP(3)R, PINK and survivin) to regulate the lipid kinase Vps-34 protein and promote formation of Beclin 1-Vps34-Vps15 core complexes, thereby inducing autophagy. In contrast, the BH3 domain of Beclin 1 is bound to, and inhibited by Bcl-2 or Bcl-XL. This interaction can be disrupted by phosphorylation of Bcl-2 and Beclin 1, or ubiquitination of Beclin 1. Interestingly, caspase-mediated cleavage of Beclin 1 promotes crosstalk between apoptosis and autophagy. Beclin 1 dysfunction has been implicated in many disorders, including cancer and neurodegeneration. Here, we summarize new findings regarding the organization and function of the Beclin 1 network in cellular homeostasis, focusing on the cross-regulation between apoptosis and autophagy.     

Molecular dynamic simulations on an inhibitor of anti-apoptotic Bcl-2 proteins for insights into its interaction mechanism for anti-cancer activity

Inhibition of normal cellular apoptosis or programed cell death is the hallmark of all cancers. Apoptotic dysregulation can result in numerous pathological conditions, such as cancers, autoimmune disorders, and neurodegeneration. Members of the BCL-2 family of proteins regulate the process of apoptosis by its promotion or inhibition and overexpression of the pro-survival anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) has been associated with tumor maintenance, growth and progression Small molecules and peptides which bind the BH3 binding groove of these proteins have been explored in the recent times for their anticancer potential. The first anticancer agents targeting this family of proteins were aimed primarily toward inhibition of Bcl-2. An elevated level of Mcl-1, despite Bcl-2 inhibition, continues to be a cause for resistance in most cancers. However, in silico exploration of Mcl-1 specific drugs and their associated mechanisms have not been clearly elucidated. In order to understand the same, we have carried out docking and molecular dynamic simulations on ABT-263 (Navitoclax), an orally active inhibitor of Bcl-2, Bcl-xL, and Bcl-w proteins; Obatoclax, a pan-Bcl-2 inhibitor as well as Maritoclax, an Mcl-1 specific inhibitor. Docking studies revealed that binding to the hydrophobic grooves is a prerequisite for action on the BCL protein and the binding mechanism and chemical space utilization dictates stability as well as specificity of the inhibitor molecular dynamic simulations showed that on binding, the α-helices of these proteins exhibited less fluctuations than loop regions, also hydrophobic contacts and hydrogen bonding were observed to be the predominant interactions in the drug-receptor complexes.

Communicated by Ramaswamy H. Sarma     

The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.     

Structural insights into the EB1-APC interaction

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms.     

Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.     

A multimeric model for murine anti-apoptotic protein Bcl-2 and structural insights for its regulation by post-translational modification.

A monomeric model for murine antiapoptotic protein Bcl-2 was constructed by comparative modeling with the software suite MPACK (EXDIS/DIAMOD/FANTOM) using human Bcl-xL as a template. The monomeric model shows that murine Bcl-2 is an all -helical protein with a central (helix 5) hydrophobic helix surrounded by amphipathic helices and an unstructured loop of 30 residues connecting helices 1 and 2. It has been previously shown that phosphorylation of Ser 70 located in this loop region regulates the anti-apoptotic activity of Bcl-2. Based on our current model, we propose that this phosphorylation may result in a conformational change that aids multimer formation. We constructed a model for the Bcl-2 homodimer based on the experimentally determined 3D structure of the Bcl-xL: Bad peptide complex. The model shows that it will require approximately a half turn in helix 2 to expose hydrophobic residues important for the formation of a multimer. Helices 5 and 6 of the monomeric subunit Bcl-2 have been proposed to form an ion-channel by associating with helices 5 and 6 of another monomeric subunit in the higher-order complex. In the multimeric model of Bcl-2, helices 5 and 6 of each subunit were placed distantly apart. From our model, we conclude that a global conformational change may be required to bring helices 5 and 6 together during ion-channel formation.Figure Hydrophobic interactions in the dimerization groove are shown. Helix 2' of monomer B interacts through residues V90, H91, L94, A97, G98, F101 and Y105 with the hydrophobic surface formed by residues in helices 3, 4, and 5 of the monomer A. Shown here is a lateral view of monomer A depicted in a surface model with hydrophobic regions in red. The backbone of the helix is shown using a neon representation in yellow and the interacting side chains are in blue.     

Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.     

Structural insights into the function of human caveolin 1

Caveolin-1 (Cav-1) is emerging as the central protein controlling caveolae formation, caveolae trafficking, and cellular signalling. In particular, it is known that Cav-1 interacts and modulates the activity of several signalling proteins through the so-called caveolin scaffolding domain. In this paper, we used a bioinformatics approach to assess the validity of some long-standing structural features of Cav-1. We could confirm the existence of a membrane spanning region of Cav-1 and highlight an interesting pattern of palmitoylated cysteine residues explaining the structural features of the Cav-1 C-terminal region. Moreover, the scaffolding domain is predicted to have a different structure than previously reported.     

Structural and biochemical insights into MLL1 core complex assembly

Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5's β-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.     

Apolipoprotein L6, induced in atherosclerotic lesions, promotes apoptosis and blocks Beclin 1-dependent autophagy in atherosclerotic cells

Inflammatory cytokine-regulated apoptosis and autophagy play pivotal roles in plaque rupture and thrombosis of atherosclerotic lesions. However, the molecular interplay between apoptosis and autophagy in vascular cells has not been investigated. Our prior study showed that human apolipoprotein L6 (ApoL6), a pro-apoptotic BH3-only member of the Bcl-2 family, was one of the downstream targets of interferon-γ (INFγ), which sensitizes atherosclerotic lesion-derived cells (LDCs) to Fas-induced apoptosis. To investigate whether ApoL6 plays a causal role in atherosclerotic apoptosis and autophagy, in this study, we demonstrate that IFNγ treatment itself strongly induces ApoL6, and ApoL6 is highly expressed and partially co-localized with activated caspase 3 in activated smooth muscle cells in atherosclerotic lesions. In addition, overexpression of ApoL6 promotes reactive oxygen species (ROS) generation, caspase activation, and subsequent apoptosis, which can be blocked by pan caspase inhibitor and ROS scavenger. Knockdown of ApoL6 expression by siApoL6 suppresses INFγ- and Fas-mediated apoptosis. Further, ApoL6 binds Bcl-X(L), one of the most abundant anti-death proteins in LDCs. Interestingly, forced ApoL6 expression in LDCs induces degradation of Beclin 1, accumulation of p62, and subsequent attenuation of LC3-II formation and translocation and thus autophagy, whereas siApoL6 treatment reverts the phenotype. Taken together, our results suggest that ApoL6 regulates both apoptosis and autophagy in SMCs. IFNγ-initiated, ApoL6-induced apoptosis in vascular cells may be an important factor causing plaque instability and a potential therapeutic target for treating atherosclerosis and cardiovascular disease.     

飞机草花蕾中Beclin1基因的克隆与表达

以入侵植物飞机草的不同时期花蕾为材料,利用RT-PCR法扩增得到了与PCD相关的类Beclin1基因的部分cDNA序列(大约700 bp),与烟草叶片中的基因序列(AY701316)同源性为95%;Northern blotting结果表明类Beclin1基因在花蕾发育中期的表达量高于初期和后期;通过DNA ladder检测表明在花蕾发育过程中伴随着PCD的发生,这些结果表明花蕾发育中期是PCD初期发生的活跃期,是绒毡层逐渐退化和花粉不断形成的过程.通过对生殖过程中的细胞程序性死亡的分子生物学研究,初步揭示了飞机草入侵过程与PCD的相互关系.     

飞机草花蕾中Beclin1基因的克隆与表达

本文以入侵植物飞机草的不同时期花蕾为材料,利用RT-PCR法扩增得到了与PCD相关的类Beclin1基因的部分cDNA序列(大约700bp),与烟草叶片中的基因序列（AY701316）同源性为95%;Northern blotting结果表明类Beclin1基因在花蕾发育中期的表达量高于初期和后期;通过DNA ladder检测表明在花蕾发育过程中伴随着PCD的发生,这些结果表明花蕾发育中期是PCD初期发生的活跃期,是绒毡层逐渐退化和花粉不断形成的过程。通过对生殖过程中的细胞程序性死亡的分子生物学研究,初步揭示了飞机草入侵过程与PCD的相互关系。     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.