Crystal structures of neuronal squid Sec1 implicate inter-domain hinge movement in the release of t-SNAREs

Sec1 molecules associate with t-SNAREs from the syntaxin family in a heterodimeric complex that plays an essential role in vesicle transport and membrane fusion. Neuronal rat n-Sec1 has an arch-shaped three-domain structure, which binds syntaxin 1a through contacts in domains 1 and 3. In both rat nSec1 and homologous squid s-Sec1, a potential effector-molecule binding-pocket is shaped by residues from domains 1 and 2 and is localized on the opposite side of the syntaxin 1a interaction site. Comparison of several crystal forms of unliganded neuronal squid Sec1 indicates a hinge region between domains 1 and 2 which allows domain 1 to rotate along a central axis. This movement could release syntaxin 1a upon interaction with a yet unspecified Sec1 effector molecule(s). The binding of an effector protein may also directly affect the conformation of the helical hairpin of domain 3, which contributes the other significant syntaxin 1a binding sites in the rat nSec1/syntaxin 1a complex structure but adopts multiple conformations in the unliganded s-Sec1 structures reported here.     

Intercellular transfer regulation of the paracrine activity of GPI-anchored Cripto-1 as a Nodal co-receptor

Cripto-1 (CR-1) is a glycosylphosphatidylinositol-anchored glycoprotein which acts as an obligate co-receptor of a TGFβ family ligand, Nodal. Previous studies have demonstrated that CR-1 functions in a paracrine fashion by a cellular mechanism which has not been fully described. This paracrine activity was observed only when CR-1 was expressed as a membrane-bound form and was abolished when CR-1 was expressed in a soluble form. In the current study, we found that there were few biochemical differences in post-translational modifications between membrane-anchored and soluble forms of CR-1. Flow cytometric analysis revealed an intercellular transfer of the membrane-bound form of CR-1 between cells. CR-1-expressing cells formed unique membrane extensions, generated more membrane fragments than control cells, and exhibited enhanced cellular adhesion. Thus, expression of CR-1 may alter the physiochemical properties of the plasma membrane resulting in an enhancement of intercellular transfer of cellular signaling components which may account for the paracrine activity of CR-1.     

Stereoselectivity of 1-aminocyclopropanecarboxylate malonyltransferase toward stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid

A malonyltransferase isolated from mungbean (Vigna radiata L.) hypocotyls catalyzed the malonylation of both 1-aminocyclopropane-1-carboxylic acid (ACC) and D-amino acids. The possibility that ACC was recognized by the enzyme as a D-amino acid was investigated by examining the efficiencies of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) serving as substrates of malonyltransferase and as inhibitors of ACC malonyltransferase. Although all four isomers were malonylated by the enzyme and competitively inhibited the malonylation of ACC to N-malonyl-ACC, (1R,2S)-AEC and (1R,2R)-AEC, both of which have an R-configuration as a D-amino acid, had lower Km and Ki values (0.1 to 0.2 mM) than their enantiomers, (1S,2R)-AEC (Km and Ki values were about 1 mM) and (1S,2S)-AEC (Km and Ki values were higher than 10 mM), which have an S-configuration as an L-amino acid. Similarly, (R)-isovaline (2-amino-2-methylbutanoic acid), which has an R-configuration as a D-amino acid, inhibited more effectively the enzymatic conversion of ACC to malonyl-ACC than did (S)-isovaline, which has an S-configuration as an L-amino acid. In mungbean hypocotyls (1R,2S)-AEC and (1R,2R)-AEC were also more efficiently converted into malonyl conjugates and more efficiently inhibited the conversion of radioactive ACC into malonyl-ACC than their enantiomers, although the differences in efficiency among stereoisomers were smaller in hypocotyls than in enzymatic reactions. These results suggest that ACC is recognized by the enzyme as a D-amino acid.     

Role of an intrinsically disordered conformation in AMPK-mediated phosphorylation of ULK1 and regulation of autophagy

Recently, it has been established that there is a direct link between adenosine monophosphate activated protein kinase (AMPK), which is an energy sensor and is activated by glucose starvation, and Unc-51-like kinase 1 (ULK1) in triggering autophagy. Proper phosphorylation of ULK1 is crucial for ULK1/AMPK association and subsequent ULK1 functions in response to nutrient deprivation. Signaling modulated via phosphorylation often involves a flexible/unstructured or an intrinsically disordered (ID) region of proteins. Structural analyses of the ULK1 protein suggest that most of its functionally important phosphorylation sites are located in an ID region. We propose that this ID nature facilitates AMPK-mediated phosphorylation of ULK1, which may provide a mechanism for ULK1 functions in response to nutrient deprivation. Understanding how an ID region of ULK1 modulates its post-translational modifications through AMPK in regulating allosteric coupling will significantly help in defining the cellular and molecular mechanisms involved in ULK1/AMPK functions and in regulation of autophagy.     

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.     

The prodomain of caspase-1 enhances Fas-mediated apoptosis through facilitation of caspase-8 activation

Caspase-1 (interleukin-1beta converting enzyme) is produced in the form of a latent precursor, which is cleaved to yield a prodomain in addition to the p20 and p10 subunits. It has been established that the (p20/p10)(2) heterotetramer processes the latent precursor of interleukin-1beta into an active form during apoptosis, but the function of the residual prodomain of caspase-1 (Pro-C1) has not been established. To evaluate the involvement of Pro-C1 in apoptosis, a Pro-C1 expression vector was transfected into the HeLa cell line, which is susceptible to Fas-mediated apoptosis. Expression of recombinant Pro-C1 in HeLa cells enhanced apoptosis mediated by Fas, but not etoposide-induced apoptosis. This enhancement of Fas-mediated apoptosis was abolished by inhibitors of caspase-8 (Ile-Glu-Thr-Asp-fluoromethyl ketone) and caspase-3 (Asp-Glu-Val-Asp-aldehyde) but was only slightly diminished by an inhibitor of caspase-1 (acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone). During apoptosis induced by an agonistic anti-Fas antibody, the activation of caspase-8 and caspase-3 was more pronounced and occurred more rapidly in HeLa/Pro-C1 cells than in the empty vector transfectant (HeLa/vec) cells; in contrast, caspase-1 was not activated in either HeLa/Pro-C1 or HeLa/vec cells. These results demonstrate an additional and novel function for caspase-1 in which Pro-C1 acts to enhance Fas-mediated apoptosis, most probably through facilitation of the activation of caspase-8.     

SOCS1基因修饰树突状细胞在肿瘤治疗中的研究进展

树突状细胞是功能最强的抗原提呈细胞,是启动、调节及维持免疫应答的核心环节,以树突状细胞为基础的肿瘤疫苗被认为是最具潜能的肿瘤免疫治疗手段。细胞因子信号通路抑制因子1（suppressor ofcytokine signaling1,SOCS1）是细胞因子信号通路抑制因子（suppressor of cytokine signaling,SOCS）家族的重要成员,广泛参与树突状细胞的发生、成熟和活化,具有负调控树突状细胞功能的重要作用。SOCS1沉默的树突状细胞能够促进自身成熟并增强其诱导的T细胞的抗肿瘤活性。现就国内外关于树突状细胞功能研究及基因修饰的肿瘤疫苗临床试验作一综述,以期对未来的研究有所帮助。     

Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of pre-formed oligomer

Apoptosis signal-regulating kinase 1 (ASK1) is a MAPKKK family member which activates c-Jun N-terminal kinase (JNK) and p38. In non-stressed cells, ASK1 exists as an inactive complex with the reduced form of thioredoxin. Oxidative stress such as hydrogen peroxide (H2O2) disrupts the ASK1-thioredoxin complex by oxidization of thioredoxin and thereby activates ASK1. The precise mechanism by which ASK1 is activated after its release from thioredoxin is unknown. Here we show that phosphorylation of Thr845 at the activation loop is essential for ASK1 to be activated by H2O2. ASK1 appears to form a silent homo-oligomer through its C-terminal coiled-coil region in non-stressed cells. Following H2O2 treatment, pre-existing ASK1 oligomer undergoes conformational change and creates a new interface within an oligomer, which ultimately leads to trans-autophosphorylation of Thr845. Thus, direct interaction via the coiled-coil region is required for self-scaffolding but not sufficient for activation of ASK1. Importantly, Thr845 of ASK1 can also be trans-phosphorylated by an unidentified Thr845 kinase in response to H2O2 treatment. We propose that this potential Thr845 kinase may be an ignition kinase that triggers Thr845 phosphorylation in oligomerized and activation-competent forms of ASK1.     

Genetic relatedness of VP1 genes of Australian and Taiwanese rotavirus isolates

Gene 1 (which encodes the viral RNA-dependent RNA polymerase, VP1) of an atypical human reassortant rotavirus strain, E210 (serotype G2P1B), is unrelated to genes 1 of standard human rotaviruses. To ascertain the origin of this gene, we determined a partial sequence and found that it exhibited greatest identity to gene 1 of a Taiwanese isolate, TE83, which is representative of G2 strains that caused an epidemic of gastroenteritis in 1993. Limited sequence identity to genes 1 of standard human and animal viruses was observed. This was confirmed by phylogenetic analysis. However, hybridization analysis using an E210 gene 1-specific probe indicated that a related gene was found among other Australian G2 isolates and in a Japanese strain isolated in the 1970s.     

ami1, an orthologue of the Aspergillus nidulans apsA gene, is involved in nuclear migration events throughout the life cycle of Podospora anserina

The Podospora anserina ami1-1 mutant was identified as a male-sterile strain. Microconidia (which act as male gametes) form, but are anucleate. Paraphysae from the perithecium beaks are also anucleate when ami1-1 is used as the female partner in a cross. Furthermore, in crosses heterozygous for ami1-1, some crozier cells are uninucleate rather than binucleate. In addition to these nuclear migration defects, which occur at the transition between syncytial and cellular states, ami1-1 causes abnormal distribution of the nuclei in both mycelial filaments and asci. Finally, an ami1-1 strain bearing information for both mating types is unable to self-fertilize. The ami1 gene is an orthologue of the Aspergillus nidulans apsA gene, which controls nuclear positioning in filaments and during conidiogenesis (at the syncytial/cellular transition). The ApsA and AMI1 proteins display 42% identity and share structural features. The apsA gene complements some ami1-1 defects: it increases the percentage of nucleate microconidia and restores self-fertility in an ami1-1 mat+ (mat-) strain. The latter effect is puzzling, since in apsA null mutants sexual reproduction is quite normal. The functional differences between the two genes are discussed with respect to their possible history in these two fungi, which are very distant in terms of evolution.     

结核分枝杆菌RD1区研究进展

RD1区是结核分枝杆菌(MTB)在长期传代过程中丢失的重要保护性抗原,RD1区仅存在于致病性分枝杆菌中,而在卡介苗(BCG)及环境分枝杆菌中缺失。RD1区基因全长9.5kb,共有9个开放读码框,分别编码9个蛋白。RD1区是MTB毒力的关键因素之一,同时RD1区存在一种新分泌表达体系,能保证ESAT6和CFP10蛋白分泌表达。RD1区蛋白有较强的免疫原性,在MTB的预防和诊断中将可能发挥巨大作用,并有可能成为筛选抗MTB药物的理想靶抗原。     

Inhibition of HIV-1 infection by an intramolecular antisense peptide to T20 in gp160

Antisense amino acids are amino acids which can be translated from the corresponding anti-codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV-1 and has been demonstrated to be a potent inhibitor of HIV-1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA-1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA-1) to T20, was shown to inhibit HIV-1(IIIB) infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. The TA-1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV-1, especially at TA-1 and in the amino acids flanking the C terminus. Although the TA-1 sites of 18 out of 30 HIV-1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV-1(MN), were not. However, TA-1L inhibited infection by HIV-1(MN), which has no antisense peptide in T20 corresponding to TA-1, although the inhibitory effect was weaker. TA-1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA-1.     

Development of a novel noncompetitive antagonist of IL-1 receptor

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.     

NOS1-mediated macrophage and endothelial cell interaction in the progression of atherosclerosis

Atherosclerosis is a chronic inflammatory disease arising due to an imbalance in lipid metabolism and maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Interactions between monocytes/macrophages and endothelial cells play an essential role in the pathogenesis of atherosclerosis. In our current study, nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) has been identified as a regulator of macrophage and endothelial cell interaction. Oxidized LDL (OxLDL) activates NOS1, which results in the expression of CD40 ligand in macrophages. OxLDL-stimulated macrophages produce some soluble factors which increase the CD40 receptor expression in endothelial cells. This increases the interaction between the macrophages and endothelial cells, which leads to an increase in the inflammatory response. Inhibition of NOS1-derived NO might serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.     

Mutation-dependent Polymorphism of Cu,Zn-Superoxide Dismutase Aggregates in the Familial Form of Amyotrophic Lateral Sclerosis

More than 100 different mutations in Cu,Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (fALS). Pathogenic mutations facilitate fibrillar aggregation of SOD1, upon which significant structural changes of SOD1 have been assumed; in general, however, a structure of protein aggregate remains obscure. Here, we have identified a protease-resistant core in wild-type as well as fALS-causing mutant SOD1 aggregates. Three different regions within an SOD1 sequence are found as building blocks for the formation of an aggregate core, and fALS-causing mutations modulate interactions among these three regions to form a distinct core, namely SOD1 aggregates exhibit mutation-dependent structural polymorphism, which further regulates biochemical properties of aggregates such as solubility. Based upon these results, we propose a new pathomechanism of fALS in which mutation-dependent structural polymorphism of SOD1 aggregates can affect disease phenotypes.     

TRPV1通道在中枢神经系统中的功能

TRPV1（transient receptor potential vanilloid 1）是在机体广泛分布的非选择性阳离子通道,能被氢离子、高温以及其它内源性和外源性配体激活.其在外周神经系统中主要参与伤害性高温的感受以及痛觉过敏等生理机制.TRPV1在中枢神经系统中功能的研究进展主要体现在突触传递,体温调节,痛觉的调制和细胞凋亡等方面.TRPV1的激活降低突触前谷氨酸的释放及增强已存在的突触后AMPA受体的作用,从而增强了突触传递效能.外周的TRPV1通过激活能够抑制血管的收缩和生热作用,从而抑制体温的升高,当TRPV1被阻断时就发生体温过高,而TRPV1体温调节的中枢作用机制可能是通过直接作用于体温调节中枢.脑干的痛觉调制环路的激活TRPV1可以引起谷氨酸盐的释放,进而激活突触后I类mGlu受体以及NMDA受体,从而起到镇痛的功能.另外近年发现TRPV1在中枢也参与呕吐、呼吸、心率及血压的调节.     

The role of arginine residues in interleukin 1 receptor binding.

Interleukin 1 (IL-1) is a family of polypeptide cytokines that plays an essential role in modulating immune and inflammatory responses. IL-1 activity is mediated by either of two distinct proteins, IL-1 alpha or IL-1 beta, both of which bind to the same receptor found on T-lymphocytes, fibroblasts and endothelial cells (Type 1 receptor). The effect of specific chemical modification of recombinant IL-1 alpha and IL-1 beta on receptor binding was examined. Modification of the proteins with phenylglyoxal, an arginine-specific reagent, resulted in the loss of Type 1 IL-1 receptor binding activity. The stoichiometry of this modification revealed that a single arginine in either IL-1 alpha or IL-1 beta is responsible for the loss of activity. Cyanogen bromide cleavage of phenylglyoxal modified IL-1 alpha and IL-1 beta, followed by sequencing of the peptides, revealed that arginine-12 in IL-1 alpha and arginine-4 in IL-1 beta, which occupy the same topology in the respective crystallographic structures, are the target of phenylglyoxal. These results suggest that an arginine residue plays an important role in ligand-receptor interaction.     

The role of molecular modeling in the design of analogues of the fungicidal natural products crocacins A and D

Extensive molecular modeling based on crystallographic data was used to aid the design of synthetic analogues of the fungicidal naturally occurring respiration inhibitors crocacins A and D, and an inhibitor binding model to the mammalian cytochrome bc(1) complex was constructed. Simplified analogues were made which showed high activity in a mitochondrial beef heart respiration assay, and which were also active against certain plant pathogens in glasshouse tests. A crystal structure was obtained of an analogue of crocacin D bound to the chicken heart cytochrome bc(1) complex, which validated the binding model and which confirmed that the crocacins are a new class of inhibitor of the cytochrome bc(1) complex.