Beclin 1对凋亡和自噬的调控作用

Beclin 1是自噬关键调控蛋白之一,参与自噬体膜形成.近期,大量研究结果指出, Beclin 1是caspase家族蛋白酶的全新底物,可被caspase剪切.剪切后的Beclin 1失去自噬调节功能,转而加剧凋亡进程.因而,Beclin 1对细胞凋亡和自噬起着重要的调控作用. 本文主要对细胞凋亡和自噬的相关性,以及Beclin 1在两通路中的调控作用进行了回顾与总结.在此基础上,进一步讨论了Beclin 1与人类疾病如肿瘤、神经系统退行性疾病的关联.最后,简要介绍了实验室常用于Beclin 1研究的工具.     

Crystal structure of Thermobifida fusca Cse1 reveals target DNA binding site

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐CRISPR‐associated (Cas) defense system is the only adaptive and inheritable immunity found in prokaryotes. The immunity is achieved through a multistep process of adaptation, expression, and interference. In the Type I‐E system, interference is mediated by the CRISPR‐associated complex for antiviral defense (Cascade), which recognizes invading double‐stranded DNA (dsDNA) through the protospacer adjacent motif (PAM) by one of the Cascade components, Cse1. Here, we report the crystal structure of Thermobifida fusca Cse1 at 3.3 Å resolution. T. fusca Cse1 reveals the chair‐like two‐domain architecture with a well‐defined flexible loop, L1, located at the larger N‐terminal domain, which was not observed in previous structures of the single Cse1 protein. Structure‐based mutagenesis analysis demonstrates that the well‐defined flexible loop and a partially conserved structural motif ([FW]‐X‐[TH]) are involved in PAM binding and recognition, respectively. Moreover, structural docking of T. fusca Cse1 into Escherichia coli Cascade cryoelectron microscopy maps, coupled with structural comparison, reveals a conserved positive patch that is contiguous with Cse2 in the Cascade complex and adjacent to the Cas3 binding site, suggesting its role in R‐loop formation/stabilization and the recruitment of Cas3 for target cleavage. Consistent with the structural observation, the introduction of alanine mutations at this positive patch abolished DNA binding activity by Cse1. Taken together, these results suggest that Cse1 is a critical Cascade component involved in Cascade assembly, dsDNA target recognition, R‐loop formation, and Cas3 recruitment for target cleavage.     

Crystal structure of a DNA binding protein from the hyperthermophilic euryarchaeon Methanococcus jannaschii

The Sac10b family consists of a group of highly conserved DNA binding proteins from both the euryarchaeotal and the crenarchaeotal branches of Archaea. The proteins have been suggested to play an architectural role in the chromosomal organization in these organisms. Previous studies have mainly focused on the Sac10b proteins from the crenarchaeota. Here, we report the 2.0 A resolution crystal structure of Mja10b from the euryarchaeon Methanococcus jannaschii. The model of Mja10b has been refined to an R-factor of 20.9%. The crystal structure of an Mja10b monomer reveals an alpha/beta structure of four beta-strands and two alpha-helices, and Mja10b assembles into a dimer via an extensive hydrophobic interface. Mja10b has a similar topology to that of its crenarchaeota counterpart Sso10b (also known as Alba). Structural comparison between the two proteins suggests that structural features such as hydrophobic inner core, acetylation sites, dimer interface, and DNA binding surface are conserved among Sac10b proteins. Structural differences between the two proteins were found in the loops. To understand the structural basis for the thermostability of Mja10b, the Mja10b structure was compared to other proteins with similar topology. Our data suggest that extensive ion-pair networks, optimized accessible surface area and the dimerization via hydrophobic interactions may contribute to the enhanced thermostability of Mja10b.     

Beclin 1 self‐association is independent of autophagy induction by amino acid deprivation and rapamycin treatment

Autophagy, a process of self‐digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti‐apoptotic proteins such as Bcl‐2 via its BH3‐like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST‐Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST‐Beclin 1. Further examination by cross linking and co‐immunoprecipitation experiments confirmed that Beclin 1 self‐interacts and that the coiled coil and the N‐terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl‐x_L, had no effect on Beclin 1 self‐interaction. Moreover, this self‐interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full‐length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein–protein interactions. J. Cell. Biochem. 110: 1262–1271, 2010. Published 2010 Wiley‐Liss, Inc.     

MicroRNA‐30a ameliorates hepatic fibrosis by inhibiting Beclin1‐mediated autophagy

We explored the role of microRNA‐30a (miR‐30a) and the mechanism involved in hepatic fibrosis. MiR‐30a overexpression was achieved by miR‐30a mimics transfection in hepatic stellate cells (HSCs) (HSC‐T6, LX‐2), and miR‐30a agomir (ago‐miR‐30a) treatment in mice. MiR‐30a levels were measured using TaqMan miRNA assay system, and the localization of miR‐30a was detected by fluorescence in situ hybridization (FISH). The interaction of miR‐30a and Beclin1 was confirmed by dual‐luciferase reporter assay. Autophagic flux was analysed using tandem mRFP‐GFP‐LC3 fluorescence microscopy, electron microscopy and Western blot of LC3‐II/I ratio. MiR‐30a was notably down‐regulated in activated HSCs and LX‐2‐exosomes induced by TGF‐β1; overexpression of miR‐30a down‐regulated extracellular matrix (ECM), such as α‐SMA, TIMP‐1, and Collagen I expression, and suppressed cell viability in HSCs. MiR‐30a was significantly down‐regulated in hepatic fibrosis mice and overexpression of miR‐30a prevented BDL‐induced fibrogenesis, concomitant with the down‐regulation of ECM. MiR‐30a inhibited HSCs autophagy and increased lipid accumulation in HSCs and in mice fibrotic hepatic tissues. MiR‐30a inhibited its downstream effector of Beclin1 by direct targeting its 3′‐UTR region. Moreover, Knock‐down of Beclin1 by small interfering RNA (siRNA) inhibited HSC autophagy and activation in LX‐2 cells. In conclusion, miR‐30a is down‐regulated in hepatic fibrosis models and its overexpression prevents liver fibrogenesis by directly suppressing Beclin1‐mediated autophagy; therefore, miR‐30a may be a new potential therapeutic target for controlling hepatic fibrosis.     

Antagonism of Beclin 1‐dependent autophagy by BCL‐2 at the endoplasmic reticulum requires NAF‐1

In addition to mitochondria, BCL‐2 is located at the endoplasmic reticulum (ER) where it is a constituent of several distinct complexes. Here, we identify the BCL‐2‐interacting protein at the ER, nutrient‐deprivation autophagy factor‐1 (NAF‐1)—a bitopic integral membrane protein whose defective expression underlies the aetiology of the neurodegenerative disorder Wolfram syndrome 2 (WFS2). NAF‐1 contains a two iron–two sulphur coordinating domain within its cytosolic region, which is necessary, but not sufficient for interaction with BCL‐2. NAF‐1 is displaced from BCL‐2 by the ER‐restricted BH3‐only protein BIK and contributes to regulation of BIK‐initiated autophagy, but not BIK‐dependent activation of caspases. Similar to BCL‐2, NAF‐1 is found in association with the inositol 1,4,5‐triphosphate receptor and is required for BCL‐2‐mediated depression of ER Ca²⁺ stores. During nutrient deprivation as a physiological stimulus of autophagy, BCL‐2 is known to function through inhibition of the autophagy effector and tumour suppressor Beclin 1. NAF‐1 is required in this pathway for BCL‐2 at the ER to functionally antagonize Beclin 1‐dependent autophagy. Thus, NAF‐1 is a BCL‐2‐associated co‐factor that targets BCL‐2 for antagonism of the autophagy pathway at the ER.     

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.     

DJ‐1 inhibits autophagy activity of prostate cancer cells by repressing JNK–Bcl2–Beclin1 signaling

The regulation of DJ‐1 on AR signaling plays an important role in the pathogenesis of prostate cancer (PCa). DJ‐1 could alter autophagy and regulate Beclin1‐involved autophagy response through JNK‐dependent pathway. JNK is known to mediate autophagy through Bcl2–Beclin1 complex. Therefore, this study aimed to investigate the significance of autophagy in DJ‐1‐modulated PCa cells. The current studies showed that DJ‐1 overexpression in LNCaP decreased LC3 transformation and autophagosome formation. However, DJ‐1 knockdown exerted the opposite effect. Moreover, DJ‐1 silencing inhibited survival and promoted death in LNCaP, which was recovered by autophagy inhibition with 3‐MA. In addition, DJ‐1 overexpression inhibited the phosphorylation of JNK and Bcl2, and the dissociation of Beclin1 and Bcl2; while the effect of silencing DJ‐1 was completely opposite. More important, JNK activated by anisimycin inhibited the proliferation and promoted death of DJ‐1‐overexpressed LNCaP while increasing LC3 transformation and LC3‐puncta formation, but these results were reversed by the decrease of Beclin1 (by spautin‐1). In contrast, when DJ‐1 was silenced, the death of LNCaP, LC3 transformation, and LC3‐puncta formation were inhibited by JNK inhibitor SP600125, which promoted cell proliferation. However, Bcl2 inhibition (by ABT737) reversed all the effects of SP600125. Our results suggested that DJ‐1 in PCa cells could promote the growth of PCa through autophagy inhibition, and JNK–Bcl2–Beclin1 signaling played an important role in it. The study provided new insights into the role of DJ‐1 in the development of PCa.     

Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non‐catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N‐formyl‐l ‐glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580–1588. © 2017 Wiley Periodicals, Inc.     

Targeting the autophagy pathway using ectopic expression of Beclin 1 in combination with rapamycin in drug-resistant v-Ha-<Emphasis Type="Italic">ras</Emphasis>-transformed NIH 3T3 cells

The effectiveness of an apoptosis-targeting therapy may be limited in tumor cells with defects in apoptosis. Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which results in autophagic cell death. In our study using multidrug-resistant v-Ha-rastransformed NIH3T3 (Ras-NIH 3T3/Mdr) cells, we demonstrated that rapamycin-induced cell death may result from 2 different mechanisms. At high rapamycin concentrations (≥ 100 nM), cell death may occur via an autophagy-dependent pathway, whereas at lower concentrations (≤ 10 nM), cell death may occur after G1-phase cell cycle arrest. This effect was accompanied by upregulation of p21^Cip1 and p27^Kip1 expression via an autophagy-independent pathway. We also tested whether inhibition of mTOR with low concentrations of rapamycin and ectopic Beclin-1 expression would further sensitize multidrug resistance (MDR)-positive cancer cells by upregulating autophagy. Rapamycin at low concentrations might be insufficient to initiate autophagosome formation in autophagy but Beclin-1 overexpression triggered additional processes downstream of mTOR during G₁ cell cycle arrest by rapamycin. Our findings suggest that these combination strategies targeting autophagic cell death may yield significant benefits for cancer patients, because lowering rapamycin concentration for cancer treatment minimizes its side effects in patients undergoing chemotherapy.     

mTOR and Beclin1: Two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury

Autophagy is the general term of lysosomal degradation of substances in cells, which is considered the key to maintaining the normal structure and function of the heart. It also has a correlation with several heart diseases, in particular, myocardial ischemia/reperfusion (I/R) injury. At the stage of myocardial ischemia, autophagy degrades nonfunctional cytoplasmic proteins providing the critical nutrients for the critical life activities, thereby suppressing cell apoptosis and necrosis. However, autophagy is likely to affect the heart negatively in the reperfusion stage. Mammalian target of rapamycin (mTOR) and Beclin1 are two vital autophagy-related molecules in myocardial I/R injury playing significant roles in different stages. In the ischemia stage, mTOR plays its roles through AMPK/mTOR and phosphoinositide 3-kinase/Akt/mTOR pathway, whereas Beclin1 plays its roles through its upregulation in the reperfusion stage. A possible interaction between mTOR and Beclin1 has been reported recently, and further studies need to be done to find the underlying interaction between the two molecules in myocardial I/R injury     

Crystal structure of Bordetella pertussis BugD solute receptor unveils the basis of ligand binding in a new family of periplasmic binding proteins

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.     

Crystal structure of a novel fold protein Gp72 from the freshwater cyanophage Mic1

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + β class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.     

Low expression of Beclin 1, associated with high Bcl-xL,predicts a malignant phenotype and poor prognosis of gastric cancer

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Crystal structure of the bacterial nucleoside transporter Tsx

Tsx is a nucleoside-specific outer membrane (OM) transporter of Gram-negative bacteria. We present crystal structures of Escherichia coli Tsx in the absence and presence of nucleosides. These structures provide a mechanism for nucleoside transport across the bacterial OM. Tsx forms a monomeric, 12-stranded beta-barrel with a long and narrow channel spanning the outer membrane. The channel, which is shaped like a keyhole, contains several distinct nucleoside-binding sites, two of which are well defined. The base moiety of the nucleoside is located in the narrow part of the keyhole, while the sugar occupies the wider opening. Pairs of aromatic residues and flanking ionizable residues are involved in nucleoside binding. Nucleoside transport presumably occurs by diffusion from one binding site to the next.     

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an alpha-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.